Methods for Identifying Functionally Related Genes and Drug Targets

ABSTRACT

The identification and evaluation of mRNA and protein targets associated with mRNP complexes and implicated in the expression of proteins involved in common physiological pathways is described. Effective targets are useful for treating a disease, condition or disorder associated with the physiological pathway.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/309,788, filed on Dec. 4, 2002, which is a continuation-in-part ofU.S. patent application Ser. No. 09/750,401, filed on Dec. 28, 2000, nowissued as U.S. Pat. No. 6,635,422, which claims the benefit of U.S.Provisional Application Ser. No. 60/173,338, filed Dec. 28, 1999, all ofwhich are hereby incorporated by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant number R01CA79907 from the National Institutes of Health. The United StatesGovernment has certain rights in this invention

FIELD OF THE INVENTION

The invention provides methods and compositions for identifying andcharacterizing functionally related gene products that are associatedwith mRNA-protein (mRNP) complexes and for characterizing cellular geneexpression. The invention also provides methods and compositions foridentifying and characterizing therapeutic targets and therapeutics.

BACKGROUND OF THE INVENTION

Most genes are regulated by a complex array of interactions, resultingin unique gene expression patterns. Such gene expression patterns varybetween different cell types, cells at different developmental stages ordifferentiation states, and cells exposed to signaling molecules,stress, infection, or other cellular condition or disorder. Efforts tounderstand the processes that regulate variant gene expression patternshave concentrated on early events in transcription regulation and inevents surrounding translation. In contrast, much less attention hasbeen paid to the role of mRNA-protein complexes (mRNP complexes) inpost-transcriptional regulatory processes, such as the regulation ofstability, localization, and translation efficiency of mRNAs. Still lessis known about the post-transcriptional processes that coordinate theexpression of functionally related genes (e.g., genes that share orparticipate in a certain function or pathway), which are oftenco-localized to particular mRNP complexes and bound to the same RNAbinding protein (RBP).

Post-transcriptional gene regulation of some mRNAs is mediated byregulatory elements or sequences that reside in both the introns andexons of pre-mRNAs, and the coding and noncoding regions of maturetranscripts. One example of such a regulatory element is the AU-richinstability element (ARE) present in the 3′-untranslated regions (UTRs)of early-response gene mRNAs, many of which encode proteins essentialfor growth and differentiation. RNA binding proteins associated withmRNP complexes bind to AREs in vitro and mediate post-transcriptionalmRNA stability and translation in vivo. However, not all mRNAs that bindto an RNA binding protein possess an ARE or other common regulatoryelement. Moreover, the mechanism(s) by which an RNA binding proteinrecognizes mRNAs that do not contain an ARE is not known.

In vitro binding assays using RNA binding proteins have shown that themRNAs that are associated with a particular RNA binding protein areoften structurally or functionally related. However, these in vitromethods do not reflect the dynamic nature of mRNA association with mRNPcomplexes in vivo, which changes in response to intra- and inter-cellular signaling events. A need therefore exists for reliable methodsfor monitoring RNA binding protein-mRNA interactions, as well as theassociation of mRNAs and proteins with mRNP complexes in vivo. The useof such methods will allow for the characterization of mRNA-proteininteractions and their functional implications, will elucidatebiological pathways, and will further allow for the identification oftherapeutic targets and therapeutics.

SUMMARY OF THE INVENTION

The invention provides methods and compositions that are used toidentify, utilize, and characterize mRNP complexes to identifyfunctionally related gene products that are coordinately expressed andassociated with a particular mRNP complex. The gene products associatedwith a particular mRNP complex are classified into biologically relevantsubsets on the basis of structural and/or functional relationships.These gene products, including mRNAs, RNA binding proteins, other mRNPcomplex-associated proteins, may participate in a particular biologicalpathway, such as an enzyme pathway, or may participate in other cellularevent or pathology, such as tumor growth, apoptosis, differentiation,aging, or cell toxicity, for example. The functionally and structurallyrelated gene products that are identified and quantified create aribonomic profile for the cell or population of cells. This ribonomicprofile provides a snapshot of the flow of genetic information at agiven time in the life of the cell or cell population, in a normal ordiseased state, or in response to an environmental influence or drug.The ribonomic profile is used as a diagnostic marker for disease orother cellular event and to rapidly identify therapeutic targets andtherapeutics that alter the expression of one or more of the mRNPcomplex-associated gene products. The identified gene productsthemselves are also used as diagnostic and therapeutic indicators.

For example, the invention provides methods for diagnosing a disease orrisk of disease, as well as monitoring a disease state, by identifyingand monitoring changes in the expression of mRNP complex-associated geneproducts in a subject's cell sample and comparing the gene expression tothat of a normal subject or other non-diseased cell sample. For example,the invention is useful for assessing the cell types present in apopulation of cells, such as in a tumor, biopsy, or body fluid, bycomparing the ribonomic profile of a cell sample to signature RNPprofiles characteristic of certain cell types. The identification ofcertain cell types is useful for diagnosing a tumor or other cellularpathology and for indicating a treatment regimen.

The invention provides useful methods for identifying a therapeutictarget by contacting a cell sample with a test compound, isolating mRNPcomplexes, and identifying an mRNP complex component whose expression isaltered in response to the compound. The therapeutic target may be anycomponent of the RNP complex or a gene or RNA encoding the component.For example, the RNAs isolated from an RNP complex may be used to probenucleic acid arrays to identify which genes are affected by the testcompound.

The invention also provides methods for assessing the efficacy of a testcompound as a therapeutic. A cell sample is contacted with a testcompound and the mRNP complexes of the cell sample are used to prepare aribonomic profile that demonstrates changes in expression of geneproducts associated with the mRNP complexes. A difference in the levelof expression of the gene products in the treated cell sample comparedto the levels in an untreated cell sample is indicative that the testcompound is a candidate therapeutic.

The invention may also be used to determine the toxicity of a testcompound and to identify genes that participate in cell death. Toxicitycan be determined by treating a cell sample with different doses of atest compound, as described above. An array containing nucleic acidsthat encode regulatory molecules, such as transcription factors, isprobed using the RNA isolated from a particular RNP complex, in order toidentify the transcription factors, RNA binding proteins, or any othertranscriptional, post-transcriptional, translational, orpost-translational regulator whose expression is altered in the presenceof specific toxicants, in order to identify downstream genes affected bychanges in these regulators.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic overview of the flow of genetic information fromthe genome to the proteome, the intermediate levels represented by theribonome and the transcriptome. The transcriptome represents the totalmRNA complement of the genome, but does not necessarily correlatedirectly with protein production. The processing, transport andtranslation of mRNA occurs in the ribonome, which represents the dynamicregulatory steps that determine the proteomic outcome.

FIG. 2A is an illustration-showing transcription of mRNAs that associatewith mRNP complexes (e.g., mRNP1, mRNP2, mRNPX). FIG. 2B is anillustration comparing arrays of total cellular mRNA, nuclear run-offmRNA, and mRNA that is bound within mRNP complexes.

FIG. 3 is a schematic outlining a strategy for the identification of newRNA binding proteins.

FIGS. 4A and 4B illustrate multiprobe RNase protection analysis of mRNAsassociated with mRNP complexes. mRNP complexes from cell lysates wereimmunoprecipitated and the pelleted RNA was extracted and quantitated byRNase protection. FIGS. 4A and 4B show examples of mMyc and mCyc-1multiprobe template sets, respectively. Lanes: (1) undigested riboprobe(slightly larger than RNase-digested product due to riboprobe plasmidtemplate); (2) total cellular RNA; (3) rabbit pre-bleed serum control;(4) mRNAs extracted from HuB mRNP complexes; (5) mRNAs extracted frompoly A-binding protein (PABP) mRNP complexes. An asterisk (*) denotesmRNA species not detected in total RNA.

FIG. 5 is a schematic outlining ribonomic profiling of RNA subsets usingDNA arrays. The mRNP complexes are isolated from two cell samples (e.g.,cells of two individuals, species, cell types, treatments, ordevelopmental stages, for example) and associated RNA poolsreverse-transcribed to make RNA probes. A DNA array of genes or cDNAs isprobed with each pool of mRNP-derived probes to generate gene expressionsubprofiles (10). Subprofiles are then compared by subtraction oraddition to generate an overall gene expression profile (20). Thesubprofiles (mRNP1, mRNP2 . . . mRNPn) of the total cell profile areshown as additive. Each stacked subprofile represents individual mRNPcomplexes within a single cell type, or can represent each individualcell's transcriptome within a complex tissue or tumor.

FIG. 6 shows the results of illustrative Example 2B, below, and showsmRNAs associated with mRNP complexes using cDNA arrays. Panels: (A)pre-bleed; (B) HuB, mRNP complexes; (C) e1F-4E mRNP complexes; (D) polyA-binding protein (PABP) mRNP complexes; (E) total cellular RNA. Anexample of the specificity of the procedure is indicated by thedifferential abundance of the mRNAs encoding β-actin and ribosomalprotein S29 among the mRNP profiles (arrows a and b, respectively).

FIG. 7 shows the results of illustrative Example 2 and shows acomparison of the mRNA profiles from HuB and HuA mRNP complexes beforeand after treatment with retinoic acid. Panels: (A) mRNAs extracted fromHuB mRNP complexes immunoprecipitated from untreated cells; (B) mRNAsextracted from HuB mRNP complexes immunoprecipitated from retinoicacid-treated cells; (C) mRNAs extracted from HuA (HuR) mRNP complexesimmunoprecipitated from untreated cells; (D) mRNAs extracted from HuAmRNP complexes immunoprecipitated from retinoic acid-treated cells; (E)total complement of mRNAs extracted from untreated cellular lysate; and(F) total complement of mRNAs extracted from retinoic acid-treatedcellular lysate.

FIG. 8 is a is a comparison of global gene expression profiling withribonomic profiling. The figure shows representative microarray spotsfrom FIG. 7 that are aligned and enlarged to compare levels of IGF-2,integrin β, cyclin D2, and HSP84 total mRNA with that associated withHuB-bound mRNAs, before and after retinoic acid treatment. Total andHuB-associated IGF-2 mRNA levels increased in response to retinoic acidtreatment. By contrast, HuB-associated integrin β, cyclin D2 and Hsp 84mRNA levels increased or decreased disproportionately to changes intheir total RNA levels after RA treatment. *, mRNAs detected only inRA-treated cells.

FIG. 9 is a schematic overview of the target discovery process using RNAbinding proteins and mRNP complexes.

FIG. 10 is a schematic overview of the data flow for analyzing andinterpreting microarray results from comparative RNA binding proteinexpression and/or mRNP complexes.

FIG. 11 is a schematic overview of the Ribonomic Analysis System (RAS™)assay compared to microarray analysis of total cellular RNA. otal RNA bymicroarray analysis

FIGS. 12A, 12B, and 12C show the results of illustrative Example 28 andshows a comparison of the expression profiles of three genes, (A)calcium channel beta 3 subunit (CACN), (B) cadherin EGF LAG seven-passG-type receptor (CELSR), and (C) muscle blind RNA binding protein (MBNL)in G11 cells, which contain a stably transfected g10-tagged HuB gene,following induction of neuronal differentiation with retinoic acid (RA)and the control condition of muscle differentiation withdimethyl-sulfoxide (DMSO). The expression of these three genes wasanalyzed by Quantitative RT-PCR during a time course of one to nine daysof treatment with either dimethyl-sulfoxide or retinoic acid.

FIGS. 13A, 13B, and 13C show the results of illustrative Example 2 andshows a comparison of the expression profiles of three genes, (A)calcium channel beta 3 subunit (CACN), (B) cadherin EGF LAG seven-passG-type receptor (CELSR), and (C) muscle blind RNA binding protein (MBNL)in the parental p19 cells following induction of neuronaldifferentiation with retinoic acid (RA) and the control condition ofmuscle differentiation with dimethyl-sulfoxide (DMSO). The expression ofthese three genes was analyzed by Quantitative RT-PCR during a timecourse of one to nine days of treatment with either DMSO or RA.

FIG. 14 is a schematic overview of the data flow for analyzing andinterpreting microarray results from toxicogenomic studies of RNAbinding protein expression profiles.

FIG. 15 depicts the use of transcriptional regulators (transcriptionfactors and RNA binding proteins) in mechanism-of-action studies.Expression profiling by microarray analysis of transcription factors andRNA binding proteins (RBPs) using the RiboChip™ microarray is useful forassessing the potential toxicity or for determining themechanism-of-action studies of drugs or therapeutics. Transcriptionfactors and RNA binding proteins represent ‘sentinel regulators’ of allgene expression changes. In the case of transcription factors,mechanism-of-action studies include studying the transcription factorbinding elements in promoter regions of regulated genes. In the case ofRNA binding proteins, the mechanism-of-action studies include studyingthe mRNA pools that are bound endogenously by particular RNA bindingproteins.

FIG. 16 is a schematic overview of the RiboTrap™ assay. An mRNA encodingan RNA binding protein (RBP1 or RBP2) of interest tagged with a ligandsuch as MSII coat protein (CP) binding site (RNA stem loops) isintroduced into a cell by transfection and expressed. The tag allows forrecovery of the mRNA with its attached RNA binding protein. A bindingpartner for the ligand such as CP antibody is used to immunoprecipitatethe tagged mRNA and its associated RNA binding proteins.

DETAILED DESCRIPTION

The present invention provides methods for mining and characterizing thecellular ribononome by identifying and measuring the mRNAs and proteinsthat are functionally co-associated with mRNA-protein (mRNP) complexes.The invention focuses on obtaining and utilizing the genetic regulatoryinformation residing along the protein biosynthetic pathway between thegenome and the proteome (FIG. 1).

The present invention identifies the components of mRNA-protein (mRNP)complexes as a valuable tool for diagnosing, monitoring, or assessingthe metabolic and disease state of cells, for identifying potentialtherapeutic targets, and for identifying and assessing the efficacy ortoxicity of potential therapeutics. Moreover, the present inventionprovides methods for identifying and characterizing structurally and/orfunctionally related gene products, to elucidate biological pathways orprocesses.

Generally, an mRNP complex consists of various components that mayinclude, but are not limited to, at least one RNA binding protein, atleast one associated or bound mRNA, at least one associated or boundprotein (i.e., an mRNP complex-associated protein), and may also consistof other associated or bound molecules (e.g., carbohydrates, lipids,vitamins, etc.). A component associates with an mRNP complex if it bindsor otherwise attaches to the mRNP complex with a Kd of about 10⁻⁶ toabout 10⁻⁹. In a preferred embodiment, the component associates with thecomplex with a Kd of about 10⁻⁷ to about 10⁻⁹. In a more preferredembodiment, the component associates with the complex with a Kd of about10⁻⁸ to about 10⁻⁹.

The associated or bound mRNAs are categorized into distinct subsetsbased on their association with a particular RNA binding protein or mRNPcomplex-associated protein. By isolating each mRNP complex in a cell,and, preferably, identifying the components of the mRNP complex and thegene precursors and gene products of those components, a ribonomic geneexpression profile for that cell can be generated. By identifying themRNA components of a cellular ribonome, the cellular transcriptome canbe broken down into a series of subprofiles that together can be used todefine the gene expression state of a cell or tissue (see FIG. 2).Ribonomic profiles will differ from cell sample to cell sample,depending on a variety of factors including, but not limited to, thedifferentiation status of the cell, the species or tissue type of thecell, the developmental stage of the cell, the pathogenicity of the cell(e.g., if the cell is infected, is expressing a deleterious gene, islacking a particular gene, is not expressing a particular gene, or isoverexpressing a particular gene), the specific ligands used to isolatethe mRNP complexes, the various conditions affecting the cell (e.g.,environmental, apoptotic or stress states, and disease or otherdisorder) and other factors known to practitioners in the art.

Isolation of mRNP Complexes

An mRNP complex is isolated from a natural biological sample such as atissue, a cell, a body fluid, an organ, or an organism. In a preferredembodiment, the biological sample is obtained from a population ofcells. The population of cells may contain a single cell type.Alternatively, the population of cells may contain a mixture ofdifferent cell types from either primary or secondary cultures or from acomplex tissue such as a tumor.

In one embodiment, the mRNP complex is isolated from a cell sample inwhich the expression of a component of an mRNP complex has been altered,e.g., induced or inhibited In another embodiment, a particular mRNPcomplex or component or precursor for one or more components of the mRNPcomplex has been introduced into the sample or has been geneticallyaltered. Introduction of the one or more mRNP complex components mayoccur by infection, transformation, or other similar methods known inthe art. In one embodiment, an expression vector expressing one or morecomponents of an mRNP complex is transfected into the cell. Suitablevectors include, but are not limited to, recombinant vectors such asplasmid vectors or viral vectors. The component is preferablyoperatively linked to appropriate promoter and/or enhancer sequences forexpression in the cell. In an embodiment of the invention, a specificcell type is engineered to contain a cell type-specific or induciblegene promoter that drives expression of an RNA binding protein. Aligand, such as an antibody that is specific for this RNA bindingprotein, may immunoprecipitate the RNA binding protein, with itsattached or associated mRNAs, from a tissue extract containing the celltype of interest. The RNAs are then identified to form the expressionprofile of that cell type or isolated for further research, as describedherein.

Alternatively, the cell sample may contain a knock out cell line orknock out organism that either does not express a component of the mRNPcomplex or expresses decreased levels of the component. Preferably, theknock out cell line or knock out organism does not express a particularRNA binding protein, an mRNA associated with the mRNA complex or RNAbinding protein, or an mRNP complex-associated protein.

In a preferred embodiment, the nucleic acid encoding the mRNP complexcomponent is tagged (e.g., a tagged RNA binding protein) in order tofacilitate the separation, observation and/or detection of thecomponents. Accessible epitopes may be used or, where the epitopes onthe components are inaccessible or obscured, epitope tags on ectopicallyexpressed recombinant proteins may be used. Suitable tags include, butare not limited to, biotin, the MS2 protein binding site sequence, theU1snRNA 70k binding site sequence, the U1snRNA A binding site sequence,the g10 binding site sequence (Novagen, Inc., Madison, Wis.), andFLAG-TAGS (Sigma Chemical, St. Louis, Mo.). For example, a transformedcell containing a transfected vector directing the expression of atagged RNA binding protein can be mixed with other cell types or can beimplanted in an animal or human subject. In an embodiment, a ligand,such as an antibody or antibody fragment, that is specific for the tagis used to immunoprecipitate the tagged RNA binding protein with itsassociated mRNAs from a tissue extract containing the transformed cell.The mRNP complexes and associated RNAs can then be identified either toform an expression profile for that cell type for further analysis.

The expression of one or more mRNP complex components may be altered bycontacting or treating the cell sample with a known or test compound.The compound may be, but is not limited to, a protein, a nucleic acid, apeptide, an antibody, an antibody fragment, a small molecule, or anenzyme. Where the compound is a nucleic acid, the nucleic acid may be anantisense nucleic acid, a ribozyme, an RNAi, an aptamer, a decoy nucleicacid, or a competitor nucleic acid. In one embodiment, the compound mayalter the expression of an mRNP complex component through competitivebinding. A compound may inhibit binding between an RNA binding proteinand an mRNA, between an RNA binding protein and an mRNPcomplex-associated protein, or between an mRNA and an mRNPcomplex-associated protein, for example. In another embodiment, the cellsample is infected with a pathogen, such as a virus, bacteria, prion,fungus, parasite, or yeast, to alter expression of one or more mRNAcomplexes.

While any method for the isolation of an mRNP complex may be used in thepresent invention, the methods disclosed in co-pending U.S. applicationSer. Nos. 09/750,401 and 10/238,306 are preferred, the disclosures ofwhich are hereby incorporated by reference. The in vivo methods forisolating an mRNP complex involve contacting a biological sample thatincludes at least one mRNP complex with a ligand that specifically bindsa component of the mRNP complex. For example, the ligand may be anantibody, a nucleic acid (e.g., an antisense, aptamer, or RNAimolecule), or any other compound or molecule that specifically binds thecomponent of the complex. In certain embodiments, the ligand is obtainedby using the serum of a subject that has a disorder known to beassociated with the production of mRNP complex-specific antibodies orproteins. Examples of these disorders include autoimmune disorders and anumber of cancers. In certain embodiments, the ligand is tagged withanother compound or molecule in order to facilitate the separation,observation or detection of the ligand. In one embodiment of theinvention, the ligand is epitope tagged, as described in the art.

In an embodiment, the mRNP complex is separated by binding the ligand(now bound to the mRNP complex) to a binding molecule that specificallybinds the ligand. The binding molecule may bind the ligand directly(e.g., a binding partner specific for the ligand), or may bind theligand indirectly (e.g., a binding partner specific for a tag on theligand). Suitable binding molecules include, but are not limited to,protein A, protein G, and streptavidin. Binding molecules may also beobtained by using the serum of a subject suffering from a disorder suchas an autoimmune disorder or cancer. In an embodiment, the ligand is anantibody that binds a component of the mRNP complex via its Fab regionand a binding molecule binds the Fc region of the antibody.

In an embodiment, the binding molecule is attached to a support (e.g., asolid support such as a bead, well, pin, plate, or column). Accordingly,the mRNP complex is attached to the support via the ligand and bindingmolecule. The mRNP complex may then be collected by removing it from thesupport (e.g., by washing or eluting it from the support using suitablesolvents and conditions that are known to a skilled artisan).

In certain embodiments of the invention, the mRNP complex is stabilizedby cross-linking prior to binding the ligand thereto. Generally,cross-linking involves covalent binding (e.g., covalently binding thecomponents of the mRNP complex together). Cross-linking may be carriedout by physical means (e.g., by heat or ultraviolet radiation), orchemical means (e.g., by contacting the complex with formaldehyde,paraformaldehyde, or other known cross-linking agents), methods of whichare known to those skilled in the art. In other embodiments, the ligandis cross-linked to the mRNP complex after binding to the mRNP complex.In additional embodiments, the binding molecule is cross-linked to theligand after binding to the ligand. In yet another embodiment, thebinding molecule is cross-linked to the support.

The methods of the invention allow for the isolation andcharacterization of a plurality of mRNP complexes simultaneously (e.g.,“en masse”). For example, a biological sample is contacted with aplurality of ligands each specific for different mRNP complexes. Aplurality of mRNP complexes from the sample bind the appropriatespecific ligands. The plurality of mRNP complexes are then separatedusing appropriate binding molecules, thereby isolating the plurality ofmRNP complexes. The mRNP complexes and the mRNAs contained within themRNP complexes are then characterized and/or identified by methodsdescribed herein and known in the art. Alternatively, the methods of theinvention are carried out on a sample numerous times and the mRNPcomplexes are characterized and identified in a sequential fashion, witheach iteration utilizing a different ligand.

Amplification of the mRNA isolated according to the methods of theinvention and/or the cDNA obtained from the mRNA is not necessary orrequired by the present invention. However, the skilled artisan maychoose to amplify the nucleic acid that is identified according to anyof the numerous nucleic acid amplification methods that are well-knownin the art (e.g., polymerase chain reaction (PCR), reverse transcriptasepolymerase chain reaction (RT-PCR), quantitative polymerase chainreaction (QT-PCR), or strand displacement analysis (SDA)).

Analysis of Isolated mRNP Complexes

The present invention provides methods for assessing the metabolic orgene expression state of a cell. Following isolation of at least onemRNP complex, the level of expression of at least one mRNA associatedwith the mRNP complex and/or at least one mRNP complex-associatedprotein is determined. In an embodiment, the level of expression of themRNA(s) or the mRNP complex-associated protein(s) on a particular mRNPcomplex provides a subprofile that is indicative of, for example, thegene expression of a subset of functionally related gene products. In anembodiment, a subset of mRNAs associated with a particular mRNP complexidentifies a ribonomic subprofile that is characteristic of a functionalRNA network or biological pathway. The collection of mRNA subsets forany particular cell or tissue sample constitutes a gene expressionprofile, and, more specifically, a ribonomic gene expression profile,for that cell or tissue. It will be appreciated that ribonomic profilesmay differ from cell to cell as described previously. Thus, theribonomic profile of a cell can be used as an identifier for the celland can be compared with profiles or subprofiles of other cells.

Accordingly, in one aspect, the present invention provides diagnosticmethods for assessing the cell types present in a sample or a populationof cells. The method involves isolating at least one mRNP complex anddetecting the expression of at least one component of the mRNP complex,wherein the at least one component is specific for a certain cell type,so that the detection of the expression of the component is indicativeof the presence of the cell type in the population of cells. Thecomponent may be specific for a certain cell type within an entiresample (e.g., tissue or organism) or within the population of cells. Thesample or population of cells may be, for example, a tumor, a tissue, acultured cell, a body fluid, an organ, a cell extract or a cell lysate.The methods of the invention may also be used to determine the celltypes present in a population of cells, where cell type may refer to thetraditional types of cells including, but not limited to, endothelial,epithelial, and smooth muscle. Alternatively, cell type, as used herein,may also refer to a class of cells derived from a particular tissue, aparticular species, a particular state of differentiation, a particulardisease state, or a particular cell cycle, etc.

In another aspect, the invention provides methods for identifying andcharacterizing functionally and/or structurally related genes and geneproducts. At least one mRNP complex is isolated, and mRNAs and/or mRNPcomplex-associated proteins are identified. The functionally relatedgene products may participate in similar pathways including, but notlimited to, enzyme pathways, pathogenesis, tumor growth, apoptosis,differentiation, aging, or cell toxicity. Genes encoding the geneproducts may also be identified according to standard methods.

An isolated mRNP complex can be examined, in part to determineexpression of its components, as a whole, or broken into its components.The MRNP complex can be separated from the ligand as a whole, or themRNA can be separated from the ligand-RNA binding protein complex,followed by separation of the RNA binding protein from the ligand.Alternatively, if the mRNA is bound to the ligand, the RNA bindingprotein can be separated from the ligand-mRNA complex, and the mRNA thenseparated from the ligand. Practitioners in the art are aware ofstandard methods of separating the components, including washing andchemical reactions. After separation, each component of an mRNP complexcan be examined and their identity, quantity, or other identifyingfactors preferably recorded (e.g., in a computer database) for futurereference.

cDNAs can be used to identify complementary mRNAs on mRNP complexespartitioned according to methods disclosed herein. cDNA microarray gridscan be used to identify mRNA subsets en masse. Microarrays are preciselyaligned grids in which each target nucleic acid (e.g., cDNA,oligonucleotide, or gene) has a position in a matrix of carefullyspotted cDNAs. Each target nucleic acid examined on a microarray has aprecise address that can be located, and the binding can be quantitated.Microarrays may be arranged in a commercially available substrate (e.g.,paper, nitrocellulose, nylon, any other type of membrane filter, chip,such as a siliconized chip, glass slide, silicone wafer, or any othersuitable solid or flexible support). In addition, mRNAs in a sample canbe identified based upon the stringency of binding and washing, aprocess known as “sequencing by hybridization.”

Alternative approaches for identifying, sequencing and/or otherwisecharacterizing the mRNAs in an mRNA subset include, but are not limitedto, differential display, phage display/analysis, SAGE (Serial Analysisof Gene Expression), and preparation of cDNA libraries from the mRNApreparation and sequencing of the members of the library.

Methods for DNA sequencing that are well known and generally availablein the art may be used to practice any of the embodiments of theinvention. The sequencing methods may employ such enzymes as the Klenowfragment of DNA polymerase I, SEQUENASE® (U.S. Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer, Boston, Mass.),thermostable T7 polymerase (Amersham, Chicago, Ill.), or combinations ofpolymerases and proofreading exonucleases such as those found in theElongase® Amplification System marketed by Gibco BRL (Invitrogen™,Carlsbad, Calif.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200) (MJ Research, Watertown, Mass.) and the ABICatalyst and 373 and 377 DNA Sequencers (Perkin Elmer, Shelton, Conn.).

In an embodiment, the methods of the invention are carried on isolatednuclei from cells (e.g., that are undergoing developmental or cell cyclechanges or that have otherwise been subjected to a cellular or anenvironmental change), performing nuclear run-off assays according toknown techniques to obtain transcribing mRNAs, and then comparing thetranscribing mRNAs with the global mRNA levels isolated from mRNPcomplexes from the same cells using cDNA microarrays. These methods thusprovide methods for distinguishing transcriptional frompost-transcriptional effects on steady state mRNA levels en masse. Forexample, FIG. 2 is a graphical illustration comparing the total cellularmRNA (the transcriptome), nuclear run-off mRNA, and mRNA that is boundwithin mRNP complexes to form a part of the ribonome. The total RNAinset depicts the total mRNA expressed in the cell (transcriptome) as amicroarray. A microarray representing a nuclear run-off experiment(lower far left) can be derived by transcription using isolated nucleiand analysis on Atlas™ arrays (BD Biosciences Clontech, Palo Alto,Calif.). As opposed to a total RNA or transcription profile that depictsRNA accumulation representing a steady-state level of mRNA, which isaffected by transcriptional and post-transcriptional events, the mRNAsdetected by nuclear run-off experiments represent only the transcriptionof a gene before the influence of post-transcriptional events. Themicroarrays representing mRNP complexes contain discrete and morelimited subsets of mRNAs than the transcriptome or nuclear run-offs. ThemRNP complex microarrays are labeled mRNP-1 through mRNP-X and depictmultiple mRNAs found in mRNP complexes isolated by using antibodiesreactive with mRNA-associated proteins.

Other methods for characterizing and identifying mRNP complex componentsinclude standard laboratory techniques such as, but not limited to,reverse transcription or quantitative PCR, RNAse protection, NorthernBlot analysis, Western blot analysis, macro- or micro-array analysis, insitu hybridization, immunofluorescence, radioimmunoassay, andimmunoprecipitation. The results obtained from these methods arecompared and contrasted in order to further characterize the functionalrelationships of the mRNA subsets and other mRNP components.

RNA binding proteins and mRNP complex-associated proteins useful in thepractice of the present invention are known in the art, or mayalternatively be identified and discovered by the methods describedherein. RNA binding proteins are involved in the control of a variety ofcellular regulatory and developmental processes, such as RNA processingand compartmentalization, mRNA splicing and transport, RNAstabilization, mRNA translation, and viral gene expression. Examples ofuseful RNA binding proteins include poly A-binding protein (“PABP”), andthe four ELAV/Hu mammalian homologues of the Drosophila ELAV RNA bindingprotein. HuA (HuR) is ubiquitously expressed while HuB, HuC and HuD (andtheir respective alternatively-spliced isoforms) are predominantly foundin neuronal tissue. HuB, HuC and HuD are also expressed as tumorcell-specific antigens in some small cell carcinomas, neuroblastomas,and medulloblastomas. All Hu proteins contain three RNA-recognitionmotifs, which confer their binding specificity for AU Rich InstabilityElements (AREs). Hu proteins bind in vitro to several ARE-containingearly response gene mRNAs including c-myc, c-fos, GM-CSF and GAP-43. Thebinding of Hu proteins to ARE-containing mRNAs can result in thestabilization and increased translatability of the mRNA transcripts. Theneuron-specific Hu proteins are one of the earliest neuronal markersproduced in teratocarcinoma cells following retinoic acid treatment toinduce neuronal differentiation.

Other exemplary RNA binding proteins are selected from the RNARecognition Motif family of cellular proteins involved in pre-mRNAprocessing. One example of such a protein is the U1A snRNP protein. Morethan 200 members of the RNA Recognition Motif superfamily have beenreported to date, the majority of which are ubiquitously expressed andconserved in phylogeny. Most have binding specificity for polyadenylatemRNA or small nuclear ribonucleic acids (e.g., U1, U2, etc.), transferRNAs, 5S or 7S RNAs. They include, but are not limited to, hnRNPproteins (A, B, C, D, E, F, G, H, 1, K, L), RNA Recognition Motifproteins CArG, DT-7, PTB, K1, K2, K3, HuD, HUC, rbp9, e1F4B, sx1, tra-2,AUBF, AUF, 32KD protein, ASF/SF2, U2AF, SC35, and other hnRNP proteins.Tissue-specific members of the RNA Recognition Motif family are lesscommon, including IMP, Bruno, AZP-RRMI, X16 which is expressed in pre-Bcells, Bj6 which is a puff-specific Drosophila protein and ELAV/Hu,which is neuron specific. RNA binding proteins and mRNPcomplex-associated proteins useful in the practice of the presentinvention include those isolated using autoimmune and cancer patientsera. A non-comprehensive list of RNA binding proteins and mRNPcomplex-associated proteins useful in the practice of the presentinvention is set forth below in Table 1.

TABLE 1 RNA Binding and mRNP Complex-Associated Proteins SLBP DAN TTPHel-N1 Hel-N2 elF-4A elF-4B elF-4G elF-4E elF-5 elF-4EBP MNK1 PABP p62KOC p90 La Sm Ro U1-70K AUF-1 RNAse-L GAPDH GRSF Ribosomal P0, P1,P2/L32 PM-Scl FMR Stauffen Crab 95 TIA-1 Upf1 RNA BP1 RNA BP2 RNA BP3CstF-50 NOVA-1 NOVA-2 CPEBP GRBP SXL SC35 U2AF I ASF/SF2 ETR-1 IMP-1IMP-2 IMP-3 ZBP LRBP-1 Barb PTB UPAmRNA BP BARB1 BARB2 GIFASBP CYP mRNABP IRE-BP p50 RHA FN mRNA BP AUF-1 GA mRNA BP Vigillin ERBP CRD-BP HuAHuB HuC HuD HnRNP A hnRNP B hnRNP C HnRNP D hnRNP E hnRNP F HnRNP GhnRNP H hnRNP K HnRNP L U2AF

The techniques described herein are used to identify new (i.e., novel orpreviously unknown) RNA binding proteins and mRNP complex-associatedproteins (FIG. 3). Thus, in one embodiment of the invention, an mRNA ofinterest (depicted in FIG. 3 as “RNA Y”) is used as “bait” to trap a newRNA binding protein. Preferably, PNA Y is first converted to a cDNAusing standard molecular biology techniques and is subsequently ligatedat the 3′ or 5′ end to a DNA tag that encodes a sequence that will binda ligand of the present invention (the ligand being illustrated asprotein “X” in FIG. 3). In other words, the tagged DNA encodes a bindingpartner of the ligand. The resulting fusion RNA is expressed in cells,where endogenous RBPs can bind and interact with RNA Y. The cells arethen lysed and cell-free extracts are prepared and contacted withProtein X, which has been immobilized on a solid support matrix. Afterincubation, Protein X and the attached RNA fusion molecule and itsassociated RNA binding proteins are washed to remove residual cellularmaterial. After washing, the newly isolated RNA binding proteins areremoved from the RNA-protein complex and identified by proteinmicrosequencing. Useful ligands include mRNP complex-specific antibodiesor proteins (e.g., obtained from a subject with an autoimmune disorderor cancer) or proteins (e.g., MSII coat protein). Useful bindingpartners include antibodies specific for the ligand.

Once partial protein sequence is obtained, the corresponding RNA bindingprotein gene may be identified from known databases of cDNA and genomicsequences or isolated from a cDNA or genomic library and sequenced.Preferably, the gene is isolated, the protein is expressed, and anantibody is generated against the recombinant RNA binding protein usingknown techniques. The antibodies are then used to recover and confirmthe identity of the endogenous RNA binding protein. Subsequently, theantibody can be used for ribonomic analysis to determine the subset ofcellular RNAs that cluster with (i.e., associate with) RNA Y. The RNAbinding protein is further tested for its ability to regulate thetranslation of the protein encoded by RNA Y, and is tested forvalidation as a drug target. Likewise, proteins encoded by the cellularRNAs that cluster with RNA Y are tested for validation as drug targets,as further described herein.

Identification of Therapeutic Targets

The invention provides methods for identifying a therapeutic target bycomparing the ribonomic subprofiles of a cell sample to the ribonomicsubprofiles of a control sample. A difference in the expression of acomponent of an mRNP complex between the two samples is indicative thatthe component is a candidate therapeutic target. The therapeutic targetmay include, but is not limited to, any component of an mRNP complex, ornucleic acid or gene product thereof. In an embodiment of the invention,the cell sample is treated with a test compound and the control samplecomprises cells that have not been treated with the test compound. Inanother embodiment, the control sample comprises cells at a differentstage in their growth cycle from the cells in the cell sample. In yetanother embodiment, the cell sample comprises a tumor cell or otherdiseased cell, and the control sample comprises a normal cell. Targetidentification includes methods known to practitioners in the art, suchas, but not limited to, the use of screening libraries, peptide phagedisplay, cDNA microchip array screening, and combinatorial chemistrytechniques known to practitioners in the art. A summary of the steps fortarget discovery is provided in FIG. 9.

Identification of Therapeutics

In another aspect, the invention provides methods for assessing theefficacy of a test compound as a therapeutic. A cell sample is contactedwith a test compound and a ribonomic profile or subprofile of the cellsample comprising the expression of at least one gene product associatedwith at least one mRNP complex is prepared. The expression levels of thegene product in the cell sample are compared to the expression levels ofthe gene product in a control sample (e.g., a cell sample that is notcontacted with a test compound). Identification of a difference inexpression of the gene product between the treated and untreated cellsamples is indicative that the test compound is a potential therapeutic.Test compounds may be, for example, nucleic acids, hormones, antibodies,antibody fragments, antigens, cytokines, growth factors, pharmacologicalagents (e.g., chemotherapeutics, carcinogenics, or other cells),chemical compositions, proteins, peptides, and/or small molecules.

In various embodiments of the invention, the therapeutic may stabilizeor destabilize the mRNA or the mRNP complex-associated protein. Inanother embodiment, the therapeutic may either inhibit translation ofthe mRNA, inhibit transport of the mRNA or the mRNP complex-associatedprotein, inhibit the binding of the RNA binding protein to the mRNA,inhibit the binding of the RNA binding protein to the mRNPcomplex-associated protein, or inhibit the binding of the mRNA to themRNP complex-associated protein, for example.

In another aspect, the invention provides methods for assessingtoxicity, potential side effects, specificity or selectivity of a testcompound, for example, by altering the concentrations or amounts of atest compound used to treat a cell sample.

In yet another aspect, the present invention provides methods forassessing or monitoring the efficacy of a therapeutic in a subject. Inaccordance with the invention, an effective amount of a therapeutic isadministered to a subject. At least one MRNP complex is isolated from acell sample from the subject, wherein altered expression of a geneproduct associated with the mRNP complex is altered by administration ofthe therapeutic. The expression of the gene product in the cell sampleafter administration of the therapeutic is compared to the expression ofthe gene product in a control sample (e.g., a second cell sampleobtained either prior to administration of the therapeutic or from anormal subject). A difference in expression between the treated and thecontrol cell samples is indicative of the efficacy of the therapeutic.The above tests can be repeated over a period of time to monitor thecontinued efficacy of the therapeutic.

Therapeutics may target over- or under-expressed proteins involved inthe etiology of a disease, disorder, or condition. Such over- orunder-expression may result in destabilization or stabilization of RNA.

Therapeutics that Destabilize mRNA

If a disease, condition or disorder is characterized by overexpressionof a protein, a therapeutic for treatment of such a condition willreduce or eliminate expression of the protein. For example, since RNAbinding proteins enhance the stability of short-lived mRNAs encodingprotooncogenes, growth factors and cytokines that contribute to cellproliferation, inhibition of RNA binding protein production mayalleviate diseases such as cancers or autoimmune diseases (e.g., bydecreasing tumor growth or inflammation, respectively). In addition, RNAbinding protein overexpression in several human tumors correlates withresistance to chemotherapy and UV irradiation. Increased stability ofc-fos, c-myc, cyclin B1 and other short-lived mRNAs in response toUV-irradiation or therapeutic drugs is well known. Accordingly,inhibition of RNA binding protein expression in these tumorsdestabilizes the mRNA in the tumors and, as a result, renders the tumorsmore responsive to cancer treatments.

In order to reduce overexpression or to cease expression of a protein ofinterest, the mRNA can be destabilized by administering an effectiveamount of a suitable test compound (e.g., an RNA binding proteininhibitor) either in vitro or in vivo. The test compound may bind mRNAso as to inhibit RNA binding protein binding to the mRNA, bind the RNAbinding protein so as to inhibit RNA binding protein binding to themRNA, bind to and destabilize the mRNP complex, and/or bind the mRNA soas to directly destabilize the mRNA, for example. Compounds that bind tothe mRNA but that do not stabilize the mRNA may inhibit the ability ofan RNA binding protein to stabilize the mRNA. If the compound bindscompetitively with an RNA binding protein, the compound can decreasemRNA stability by inhibiting the RNA binding protein's ability to bindthe mRNA.

Alternatively, the test compound may inhibit RNA binding protein or mRNAexpression.

Effective test compounds (e.g., RNA binding protein inhibitors) can bereadily determined by screening compounds for their ability to interferewith the production of RNA binding protein or their ability to inhibitthe binding to, and/or stabilization of, mRNA, for example, by methodsdescribed herein. Compounds that function by inhibiting RNA bindingprotein or mRNA production can be identified by exposing cells thatexpress the RNA binding protein or mRNA of interest and monitoring thelevels of RNA binding protein or mRNA, respectively. Compounds thatfunction by inhibiting the stabilizing effect of RNA binding protein onmRNA can be identified by combining RNA binding protein and an mRNA thatwould otherwise be stabilized, adding compounds to be evaluated as RNAbinding protein inhibitors, and monitoring the binding affinity of RNAbinding protein and the mRNA. Compounds that increase or decrease thebinding affinity of RNA binding protein and the mRNA can be readilydetermined by art known methods.

Therapeutics that Stabilize mRNA

If a disease, condition or disorder is characterized by underexpressionof an mRNA stabilizing protein, a therapeutic for treatment of such amedical condition may operate by stabilizing the mRNA associated withthe underexpressed protein. Accordingly, mRNA may be stabilized byadministering an effective amount of a compound, either in vitro or invivo. The compound may possess a similar binding ability and stabilizingeffect as the RNA binding protein or, may promote the RNA bindingprotein's ability to stabilize mRNA, and/or may promote the productionof the stabilizing RNA binding protein or the mRNA of interest. Such acompound may be referred to as an RNA binding protein inducer and mayoperate by interacting with the mRNA, the RNA binding protein or both.Alternatively, mRNA can be stabilized by administering an effectiveamount of a suitable RNA binding protein that possesses the necessarymRNA stabilizing effect.

Compounds that increase RNA binding protein production can be identifiedby initially exposing cells that express the RNA binding protein topotential inducers and, monitoring the levels of the RNA bindingprotein, in accordance with the methods described above. If the level ofRNA binding protein expression increases, the compound is an RNA bindingprotein inducer. Compounds that inhibit RNA binding protein binding tomRNA, but which bind and stabilize mRNA, can be identified by methodsdisclosed herein. A skilled practitioner may combine RNA binding proteinand an mRNA that would otherwise be stabilized, add a compound, andmonitor the binding affinity of the RNA binding protein and the mRNA.Compounds that increase or decrease the binding affinity of an RNAbinding protein and the mRNA can be readily determined by evaluating thebinding affinity of the RNA binding protein to the mRNA after exposureto the compound, as described herein. By monitoring the concentration ofmRNA over time, those compounds which bind to the mRNA can then beassayed for their ability to stabilize mRNA.

Antibody Preparation

Antibodies and fragments thereof that bind to mRNP complexes aregenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab fragments, and fragments produced by a Fab expressionlibrary. Antibodies and fragments thereof may also be generated usingantibody phage expression display techniques, which are known in theart.

For the production of antibodies, various hosts including, but notlimited to, goats, rabbits, rats, mice, and humans are immunized byinjection with the mRNP complex or any fragment or component thereofthat has immunogenic properties. Depending on the host species, anadjuvant is used to increase the immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (BacilliCalmette-Guerin) and Corynebacterium parvum are preferable.

Monoclonal antibodies to the components of the mRNP complex are preparedusing any technique that provides for the production of antibodymolecules by a cultured cell line. These include, but are not limitedto, the hybridoma technique, the human B-cell hybridoma technique, andthe EBV-hybridoma technique. Generally, an animal is immunized with themRNP complex or immunogenic fragment(s) or conjugate(s) thereof.Lymphoid cells (e.g., splenic lymphocytes) are then obtained from theimmunized animal and fused with immortalized cells (e.g., myeloma orheteromycloma) to produce hybrid cells. The hybrid cells are screened toidentify those which produce the desired antibody.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as is known in the art.

Antibody fragments that contain specific binding sites for mRNPcomplexes may also be generated. For example, such fragments include,but are not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries are constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity.

Various immunoassays are used to identify antibodies having the desiredspecificity for the MRNP complex. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between the component of the mRNP complex and its specificantibody. An immunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes is preferred, but a competitive binding assaymay also be employed.

The invention provides kits containing columns in which antibodies tovarious mRNP complexes or components thereof (e.g., antibodies to RNAbinding proteins) are immobilized. The antibodies may be conjugated to asupport suitable for a diagnostic assay (e.g., a solid support such asbeads, plates, slides or wells formed from materials such as latex orpolystyrene) in accordance with known techniques. Antibodies maylikewise be conjugated to detectable groups such as radiolabels (e.g.,³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescent labels (e.g., fluorescein) in accordancewith known techniques. Such devices preferably include at least onereagent specific for detecting the binding between an antibody and theRNA binding protein. The reagents may also include ancillary agents suchas buffering agents and protein stabilizing agents (e.g.,polysaccharides and the like). The device may further include, wherenecessary, agents for reducing background interference in a test,control reagents, apparatus for conducting a test, and the like. Thedevice may be packaged in any suitable manner, typically with allelements in a single container along with a sheet of printedinstructions for carrying out the test.

Antibodies raised against an mRNP complex can be conjugated to a drug.Upon administration to a patient, the antibody will bind to the mRNPcomplex so as to deliver a relatively high concentration of the drug tothe desired tissue or organ. In one embodiment, an antibody isconjugated to an anti-cancer drug, including, but not limited to, anantifolate, an anti-tumor antibiotics and other tumor-treatingcompounds.

Antibodies that bind to the mRNP complex can also be covalently orionically coupled to various markers, and used to detect the presence oftumors. By administering a suitable amount of the marker-coupledantibody to a patient, allowing the antibody to bind the mRNP complex ator around a tumor site, and detecting the marker, the presence of tumorscan be detected. Suitable markers are well known in the art, andinclude, but are not limited to, radioisotopic labels, fluorescentlabels and the like. Suitable detection methods for these markers arealso well known in the art and include, but are not limited to, positronemission tomography, autoradiography, flow cytometry, radioreceptorbinding assays, and immunohistochemistry.

High Throughput Screening Methods for Libraries of Compounds

In an embodiment of the invention, high throughput screening assays andcompetitive binding assays are used to identify compounds that bind toan mRNP complex or component thereof from combinatorial libraries ofcompounds (e.g., phage display peptide libraries, small moleculelibraries and oligonucleotide libraries).

In one embodiment, an mRNP component, catalytic or immunogenic fragmentthereof, or oligopeptide thereof, can be used to screen libraries ofcompounds in any of a variety of drug screening techniques. An exemplarytechnique is described in published PCT application WO84/03584, herebyincorporated by reference. The fragment employed in such screening canbe free in solution, affixed to a support (e.g., solid support), borneon a cell surface, or located intracellularly.

The SELEX method, described in U.S. Pat. No. 5,270,163 (Gold et al.),hereby incorporated by reference, is used to screen oligonucleotidelibraries for compounds that have suitable binding properties. Inaccordance with the SELEX method, a candidate mixture of single strandednucleic acids with regions of randomized sequence can be contacted withthe mRNP complex. Those nucleic acids having an increased affinity tothe mRNP complex can be partitioned and amplified so as to yield aligand enriched mixture.

Phage display technology is used to screen peptide phage displaylibraries to identify peptides that bind to an mRNP complex or componentthereof. Methods for preparing libraries containing diverse populationsof various types of molecules such as peptides, polypeptides, proteins,and fragments thereof are known in the art and are commerciallyavailable.

A library of phage displaying potential binding peptides is incubatedwith an mRNP complex to select clones encoding recombinant peptides thatspecifically bind the mRNP complex or components thereof. After at leastone round of biopanning (binding to the mRNP complex), the phage DNA isamplified and sequenced, thereby providing the sequence for thedisplayed binding peptides. Briefly, the target, an mRNP complex, can becoated overnight onto tissue culture plates and incubated in ahumidified container. In a first round of panning, approximately 2×10¹¹phage can be incubated on the protein-coated plate for 60 minutes atroom temperature while rocking gently. The plates are then washed usingstandard wash solutions. The binding phage can then be collected andamplified following elution using the target protein. Secondary andtertiary pannings can be performed as necessary. Following the lastscreening, individual colonies of phage-infected bacteria can be pickedat random, the phage DNA isolated and subjected to automated dideoxysequencing. The sequence of the displayed peptides can be deduced fromthe DNA sequence.

The biological activity of compounds can be evaluated using in vitroassays known to those skilled in the art (e.g., protein synthesis assaysor tumor cell proliferation assays). Alternatively, the biologicalactivity of the compounds are evaluated in vivo. Various compounds,including antibodies, can bind to mRNP complexes and components thereofwith varying effects on mRNA stability. The activity of the compoundsonce bound can be readily determined using the assays described herein.

Binding assays include cell-free assays in which an RNA binding proteinand an mRNA are incubated with a labeled test compound. Followingincubation, the mRNA, free or bound to a test compound, can be separatedfrom unbound test compound using any of a variety of techniques known inthe art. The amount of test compound bound to an mRNP complex orcomponent thereof is then determined, using detection techniques knownin the art.

Alternatively, the binding assay is a cell-free competition bindingassay. In such assays, mRNA is incubated with labeled RNA bindingprotein. A test compound is added to the reaction and assayed for itsability to compete with the RNA binding protein for binding to the mRNA.Free labeled RNA binding protein can be separated from bound RNA bindingprotein. By subsequently determining the amount of bound RNA bindingprotein, the ability of the test compound to compete for mRNA bindingcan be assessed. This assay can be formatted to facilitate screening oflarge numbers of test compounds by linking the RNA binding protein orthe mRNA to a support so that it can be readily washed free of unboundreactants. A plastic support (e.g., a plastic plate such as a 96 welldish) is preferred. The RNA binding protein and mRNA suitable for use inthe cell-free assays described herein can be isolated from naturalsources (e.g., membrane preparations) or prepared recombinantly orchemically. The RNA binding protein can be prepared as a fusion proteinusing, for example, known recombinant techniques. Preferred fusionproteins include, but are not limited to, a glutathione-S-transferase(GST) moiety, a green fluorescent protein (GFP) moiety that is usefulfor cellular localization studies or a His tag that is useful foraffinity purification.

A competitive binding assay may also be cell-based. Accordingly, acompound, preferably labeled, known to bind an mRNP complex or componentthereof, is incubated with the mRNP complex or component thereof in thepresence and absence of a test compound. By comparing the amount ofknown test compound associated with cells incubated in the presence ofthe test compound with that of cells incubated in the absence of thetest compound, the affinity of the test compound for the RNA bindingprotein, mRNA, and/or complex thereof can be determined. Cellproliferation can be monitored by measuring the uptake into cellularnucleic acids of labeled bases (e.g., radioactively, such as ³H, SiC, or¹⁴C; fluorescently, such as CYQUANT (Molecular Probes); orcalorimetrically such as rdU (Boehringer Mannheim) or MTS (Promega)) asknown in the art. Cytosolic/cytoplasmic pH determinations can be madewith a digital imaging microscope using substrates such asbis(carboxyethyl)-carbonyl fluorescein (BCECF) (Molecular Probes, Inc.,Eugene, Oreg.).

Other types of assays that can be carried out to determine the effect ofa test compound on RNA binding protein binding to mRNA include, but arenot limited to, the Lewis Lung Carcinoma assay and extracellularmigration assays such as the Boyden Chamber assay.

Accordingly, the methods permit the screening of compounds for theirability to modulate the effect of an RNA binding protein on the bindingof and stability of mRNA. Using the assays described herein, compoundscapable of binding to mRNA and modulating the effects on those cellularbioactivities resulting from mRNA stability and correlated proteinsynthesis are identified. The compounds identified in accordance withthe above assays are formulated as therapeutic compositions.

Diagnosing and Monitoring Disease

In another aspect, the invention provides methods for diagnosing adisease or risk of a disease in a subject. A ribonomic profile from asubject's cell sample is prepared and at least one mRNP complex isanalyzed. The expression of at least one gene product, for which alteredexpression is indicative of a disease or risk of disease, is determined.The gene product may be an RNA binding protein, an mRNA, an mRNPcomplex-associated protein or other gene product bound to or associatedwith the mRNP complex. The expression of the gene product in the cellsample is compared to the expression of the gene product in a controlsample. The control sample may be either a sample of normal cells or asecond cell sample from the subject. Alternatively, the control sampleis a positive control from a diseased and/or normal individual. Byobserving the relative expression of the gene product in the cell samplecompared to the control sample, the presence of a disease or risk ofdisease can be determined.

In another aspect, the invention discloses a method for monitoring adisease state in a subject. At least one mRNP complex is isolated from adiseased subject's cell sample, wherein the mRNP complex has at leastone gene product that is associated with the disease. The expression ofthe gene product in the subject's cell sample is compared to theexpression of the gene product in a control sample. The identificationof a difference in the expression of the gene product in the diseasedsubject cell sample compared to the expression of the gene product inthe control sample is indicative of a change in the disease state of thesubject. For example, a decrease in the production of a tumor relatedantigen or its mRNA is indicative of decreased tumor load or remission;by contrast, an increase in expression of the tumor antigen isindicative of aggressive tumor growth. Such monitoring during drugtreatment provides information about the effectiveness of the subject'sdrug regimen, and may indicate when a particular regimen is not, or isno longer, effective for treating the disease or condition. The controlsample may be a second cell sample from the subject, preferably,obtained when the subject is free of one or more symptoms of thedisease. Alternatively, the control sample is from a normal subject orother normal cell sample.

In summary, the present invention provides powerful in vivo methods fordetermining the ribonomic profile of a cell and detecting changes in theribonomic profile. The invention has numerous uses, including, but notlimited to, monitoring cell development or growth, monitoring a cellstate, and monitoring perturbations of a biological system such asdisease, condition or disorder. The invention further provides methodsfor diagnosing a disease, condition, or disorder and determiningappropriate treatment regimens. The invention also is useful fordistinguishing ribonomic profiles among organisms such as plant, fungal,bacterial, viral, protozoan, or animal species.

The present invention can be used to discriminate betweentranscriptional and post-transcriptional contributions to geneexpression and to track the movement of RNAs through mRNP complexes,including the interactions of combinations of proteins with RNAs in mRNPcomplexes. Accordingly, the present invention can be used to study theregulation of RNA stability. The present invention can be used toinvestigate the activation of translation of mRNAs as single or multiplespecies by tracking the recruitment of mRNAs to active polysomes,measuring the sequential, ordered expression of mRNAs such as mRNAs thatencode transcription factors or RNA binding proteins, and measuring thesimultaneous, coordinate expression of multiple mRNAs. The presentinvention can also be used to determine the transacting functions ofRNAs themselves upon contacting other cellular components. These andnumerous other uses will be made apparent to the skilled artisan uponstudy of the present specification and claims.

The contents of all cited references (including literature references,issued patents, and patent applications) as cited through out thisapplication are hereby expressly incorporated by reference. Thefollowing Examples are set forth to illustrate the present invention,and are not to be construed as limiting thereof.

EXEMPLIFICATION Example 1 RNase Protection in a Multiprobe System

A multiprobe RNase protection assay was used to rapidly optimize theimmuno-precipitation of several endogenous mRNP complexes containingdifferent RNA binding proteins. In the multiprobe system, many mRNAsassociated with an mRNP complex can be assayed in a single lane of apolyacrylamide gel.

Cell Culture and Transformation. Murine P19 embryonal carcinoma cellswere obtained from the American Type Culture Collection (ATCC, Manassas,Va.) and maintained in monolayer culture using α-Minimum EssentialMedium Eagle (MEM) without phenol red (Gibco BRL 41061-0291)(Invitrogen, Carlsbad, Calif.) supplemented with 7.5% Bovine Calf Serum(BCS), 2.5% Fetal Bovine Serun (FBS) (Hyclone, Logan, Vt.) and 100 U ofPenicillin/Streptomycin. Cells were grown in tissue culture flasks orplates that had been pre-coated with 0.1% gelatin (Sigma Chemicals, St.Louis, Mo.) and removed prior to use. Monolayer cell cultures weremaintained in 5% CO₂ at 37° C.

P19 cells were stably transfected with a SV40 promoter-drivenpAlpha2-gene10-HuB plasmid that ectopically expressed a gene 10-taggedneuron-specific HuB protein termed Hel-N2. The expression of thetransfected plasmid was maintained by supplementing the medium with 0.2mg/ml G418 (Sigma Chemicals, St. Louis, Mo.). Although the constructlacks thirteen amino acids from the hinge region connecting the RNArecognition motifs 2 and 3 of Hel-N1, the RNA recognition motifs areotherwise identical and in vitro binding experiments have indicated nodifferences in the AU-rich RNA binding properties of Hel-N1 and Hel-N2.

Antibodies. Monoclonal anti-gene 10 (g10) antibodies were producedaccording to standard techniques as previously described. Polyclonalsera reactive with HuA were produced according to standard techniques aspreviously described. Antibodies reactive with Poly A-binding protein(PABP) were obtained from McGill University (Canada).

Preparation of Cell Free Extracts. Cells were Removed from TissueCulture Plates with a rubber scraper and washed with cold phosphatebuffered saline (PBS). The cells were resuspended in approximately twopellet volumes of polysome lysis buffer (PLB) containing 100 mM KCl, 5mM MgCl₂, 10 mM HEPES pH 7.0, and 0.5% NP-40 with 1 mM Dithiothrietol(DTT), 100 U/mL RNase OUT (Gibco BRL, Invitrogen Corp., Carlsbad,Calif.), 0.2% vanadyl ribonucleoside complex (VRC) (Gibco BRL,Invitrogen Corp., Carlsbad, Calif.), 0.2 mM Phenylmethylsulfonylfluoride(PMSF), 1 mg/mL pepstatin A, 5 mg/mL pepstatin, and 20 mg/mL leupeptinadded fresh at the time of use. The cell lysate was frozen and stored at−100° C. At the time of use, the cell lysate was thawed and centrifugedat 12,000 rpm in a tabletop microfuge for 10 min at 4° C. Thesupernatant was removed and centrifuged a second time at 16,000 rpm in atabletop microfuge for 5 min at 4° C. before being stored on ice orrefrozen at −100° C. The mRNP containing cell lysate containedapproximately 30-50 mg/mL total protein.

Immunoprecipitation. Protein A sepharose beads (Sigma Biochemicals, St.Louis, Mo.) were swollen 1:5 v/v in NT2 buffer (50 mM Tris pH 7.4, 150mM NaCl, 1 mM MgCl₂, and 0.05% NP-40) and supplemented with 5% BSA A 300μL aliquot of the 1:5 v/v pre-swollen. Protein A beads were incubatedovernight at 4° C. with excess immunoprecipitation antibody (typically5-20 μL, depending on the antibody). The antibody-coated Protein A beadswere washed 5 times with ice-cold NT2 buffer and resuspended in 900 μLof NT2 buffer supplemented with 100 U/mL RNase OUT, 0.2%Vanady/Ribonucleoside Complexes, 1 mM DTT, and 20 mM ethylenediaminetetracetic acid (EDTA). The beads were briefly vortexed and 100μL of the mRNP lysate was added. The beads were immediately centrifugedand a 100 μL aliquot was removed to represent total cellular RNA(essentially one-tenth the quantity of lysate used in the mRNPimmunoprecipitations). The immunoprecipitation reaction and an aliquotremoved to represent total cellular RNA were tumbled at room temperaturefor a time period of from zero minutes to two hours. Followingappropriate incubation, the Protein A beads were washed four times withice-cold NT2 buffer followed by two washes with NT2 buffer supplementedwith 1 M urea. The washed beads were resuspended in 100 μL NT2 buffersupplemented with 0.1% sodium dodecyl sulphate (SDS) and 30 μgproteinase K and incubated for 30 minutes in a 55° C. water bath.Following proteinase K digestion, immunoprecipitated RNA was isolatedwith two phenol/chloroform/isoamyl alcohol extractions and ethanolprecipitated.

RNase Protection Assays. mRNP complexes were immunoprecipitated from thecell lysate and the bound RNA extracted and assayed by RNase protectionusing the PharMingen Riboquant assay (Phaminoen, San Diego, Calif.)according to the manufacturer's instructions (45014K). Briefly,extracted RNA was hybridized with excess ³²P-labeled riboprobesgenerated from templates specific for mRNAs encoding L32,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), several murineMyc-related proteins (template set 45356P) and cyclins (template set45620P). Non-duplexed RNA was digested by treatment with RNase A+T1. Theresulting fragments were resolved by denaturing polyacrylamide/urea gelelectrophoresis. Because the length of the riboprobe for each mRNAspecies was a unique size, all detectable mRNA species in a sample couldbe resolved in a single gel lane. Protected riboprobe fragments werevisualized on a phosphoimaging screen (Molecular Dynamics, Sunnyvale,Calif.) after 24 hours of exposure. Phosphoimages were scanned using theMolecular Dynamics Storm 860 System at 100 micron resolution andanalyzed using Molecular Dynamics IMAGEQUANT™ Software (V 1.1)(Molecular Dynamics, Sunnyvale, Calif.).

Results. FIG. 4 shows an immunoprecipitation of HuB and Poly-A bindingprotein (PABP) mRNP complexes from extracts of murine P19 cells stablytransfected with g10-HuB cDNA. No mRNAs were detected in pelletsimmunoprecipitated with polyclonal pre-bleed rabbit sera (FIGS. 4A and4B, lane 3), or with any other rabbit, mouse, and normal human seratested with this assay (data not shown). The profiles of mRNAsassociated with HuB mRNP complexes included n-myc, 1-mfc, b-myc, max andcyclins A2, B1, C, D1, and D2, but not sin3, cyclin D3, cyclin B2, L32or GAPDH mRNAs (FIGS. 4A and 4B, lane 4). In contrast, the profiles ofmRNAs extracted from PABP mnRNP complexes resembled the profiles oftotal RNA, but showed enriched levels of L32 and GAPDH and decreasedlevels of sin3 mRNA (FIGS. 4A and 4B, lane 5). These results areconsistent with the postulated role for Hu proteins in regulatingpost-transcriptional gene expression during cell growth anddifferentiation.

Example 2 Identification of mRNA Subsets Associated with RNA BindingProteins En Masse Using cDNA Arrays

A cDNA array (FIG. 5) was used to detect an mRNA subset withoutamplification or iterative selection.

Antibodies. Monoclonal anti-gene 10 (g10) antibodies and polyclonal serareactive with the proteins were produced as previously described.Antibody against 5′ cap binding protein (e1F-4E) was obtained fromTransduction Laboratories (San Diego, Calif.). Antibodies reactive withPoly A-binding protein (PABP) were obtained from McGill University(Canada).

Cell Culture and Differentiation. Transgenic cells were prepared asdescribed in Example 1. Cells were treated with retinoic acid (RA) toinduce neuronal differentiation as follows: 5×10⁵ p19 cells were placedon a 60 mm petri dish (Fisher Scientific, Pittsburgh, Pa., Number8-757-13A) with 0.5 μM RA (Sigma Chemicals, St. Louis, Mo., NumberR2625). After two days, 25% of the cells that had formed into clumpswere removed, placed in new petri dishes, and supplemented with freshmedium and RA. After two days, cell aggregates were washed once withphosphate-buffered saline (PBS) and trypsinized. The cells were thenplated into two 100 mm gelatin-coated tissue culture plates. Cells wereharvested after an additional four days. The RA-treated HuB (Hel-N2)stably transfected P19 cells grew neurites and displayed characteristicneuronal markers and morphology, but did not terminally differentiateand remained susceptible to killing with mitotic inhibitors. Cell-freeextracts and immunoprecipitations were obtained as described in Example1.

cDNA Array Analysis. cDNA array analysis was performed using ATLAS™Mouse Arrays (Clontech, Inc., Palo Alto, Calif.) that contain a total of597 cDNA segments spotted in duplicate, side-by-side on a nylonmembrane. Probing of cDNA arrays was performed as described in theClontech (Palo Alto, Calif.) ATLAS™ cDNA Expression Arrays User's Manual(PT3140-1). Briefly, RNA was extracted from HuB stably transfected P19embryonal carcinoma cells and used to produce reverse transcribedprobes. A pooled set of primers, complementary to the genes representedon the array, was used for the reverse transcription probe synthesis,which was radiolabeled with ³²Pα-dATP. The radiolabeled probe waspurified by passage over CHROMA SPIN™-200 columns (Clontech, Inc., PaloAlto, Calif.) and incubated overnight with an array membrane usingEXPRESSHYB™ hybridization solution (Clontech, Inc., Palo Alto, Calif.).Following hybridization, the array membrane was washed and visualized ona phosphorimaging screen (Molecular Dynamics, Sunnyvale, Calif.).

Phosphorimages were scanned using the Molecular Dynamics STORM 860System at 100 micron resolution and stored as files. Images wereanalyzed using ATLASIMAGE™ 1.0 and 1.01 software (Clontech, Inc., PaloAlto, Calif.). The signal for any given gene was calculated as theaverage of the signals at two duplicate cDNA spots. As described in theATLASIMAGE™ 1.0 software manual (Clontech, Inc., Palo Alto, Calif.), adefault external background setting was used in conjunction with abackground-based signal threshold to determine gene signal significance.The signal for a gene was considered significantly above background ifits adjusted intensity (total signal minus background) was more thantwo-fold the background signal. Comparisons of multiple cDNA arrayimages were performed using an average of all the gene signals on thearray (global normalization) to normalize the signal intensity betweenarrays. Changes in the mRNA profile of HuB mRNP complexes in response toRA treatment were considered significant if they were four-fold greater(twice the stringency typically used for establishing significance of agene expression change). cDNA array images and overlays were preparedusing ADOBE PHOTOSHOP® 5.0.2 (San Jose, Calif., USA).

Results. After assessing the overall gene expression profile of the HuBtransfected P19 cells (the transcriptome), HuB and PABP mRNA complexes,as well as e1F-4E mRNP complexes, were separately immunoprecipitated andcaptured mRNAs were identified on cDNA arrays. The initial alignment ofthese arrays was facilitated by spiking the hybridization reaction withradiolabeled lambda phage markers that hybridized with six DNA spots onthe bottom of the array membrane. Once the alignment register wasestablished, subsequent blots did not require the use of spiked lambdamarkers for orientation.

Arrays generated from immunoprecipitations with rabbit pre-bleed serawere essentially blank, with the exception of the spiked lambda markersobserved at the bottom of the array (FIG. 6A). Immunoprecipitated HuBmRNP complex and e1F-4E mRNP complexes contained slightly more than 10%of the mRNAs detected in total cell RNA, but differed considerably fromone another (FIGS. 6B, 6C, and 6E).

Like HuB and e1F-4E, PABP has been implicated in facilitating mRNAstabilization and translation. Not surprisingly, PABP-associated mRNPcomplexes contained many more detectable mRNAs than those observed inthe HuB or e1F-4E mRNP complexes (FIG. 6D). As expected, the profile ofthe mRNAs in the PABP mRNP complexes from these cells closely resembledthat of the transcriptome. However, as was seen for HuB and e1F-4E mRNPcomplexes, some mRNAs were enriched or depleted in the PABP-mRNPcomplexes as compared to the total RNA (FIGS. 6D and 6E). The profilesand relative abundance of mRNAs detected in these mRNP complexes werehighly reproducible, but the absolute number of mRNA species detectableon the phosphorimages occasionally varied as a result of differences inthe specific activity of the probe.

Because the cDNA arrays derived using total RNA were generated usingone-tenth the quantity of lysate used for mRNP complexesimmunoprecipitations, a comparison of the absolute quantities of eachmRNA detected in mRNP complexes with those observed in the total RNA wasnot conducted. A more accurate result was obtained by comparing therelative abundance of each mRNA species to each other within eachmicroarray. For example, the relative abundance of the mRNA encodingβ-actin and ribosomal protein S29 (FIG. 6, arrows a and b, respectively)is approximately equal in total cellular RNA, but varied dramaticallyamong each of the mRNP complexes. These findings indicated that the mRNAprofiles detected for HuB, e1F-4E and PABP mRNP complexes are distinctfrom each other and from those of the transcriptome.

Example 3 Alterations in mRNP Complexes in Response to Retinoic Acid

Because HuB is predominantly a neuronal protein believed to play a rolein regulating neuronal differentiation, studies were conducted toinvestigate whether the mRNA population found in HuB mRNP complexeschanges in response to RA, a chemical inducer of neuronaldifferentiation. HuB-transfected P19 cells were treated with RA toinduce the onset of neuronal differentiation, HuB mRNP complexes wereimmunoprecipitated, and then associated mRNAs were identified on cDNAarrays as described in Examples 1 and 2. Comparison of the mRNA profilesextracted from the HuB mRNP complexes before and after RA treatmentrevealed that eighteen mRNAs were either exclusively present or greatlyenriched (four-fold or greater) in RA-treated HuB mRNPs (FIGS. 7A and7B). In addition, three mRNAs (T-lymphocyte activated protein,DNA-binding protein SATB1, and HSP84) decreased in abundance byfour-fold or greater in response to RA treatment (FIGS. 7A and 7B) Todetermine if the changes observed in the mRNA profile of the HuB mRNPcomplexes were unique, the mRNA complexes for the ubiquitously expressedELAV family member HuA (HuR) was immunoprecipitated from these RAtreated cells. Although there were a few changes to the HuA mRNA profilefollowing treatment with the RA, they were minor in comparison with theHuB mRNA profile (FIGS. 7C and 7D).

The changes in the HuB-associated mRNA profile in response to RAtreatment did not merely reflect changes in the total cellular mRNA(FIGS. 7E and 7F). However, in some cases, changes in the HuB mRNAprofile reflected global changes in the total RNA profile. Numerousexamples of differentially-enriched or depleted mRNAs detected in HuBmRNP complexes are evident by comparing FIGS. 7A and 7B to FIGS. 7E and7F. For comparative purposes, selected examples of mRNAs thatdemonstrate differences between the total RNA profile in comparison withHuB-bound mRNAs before and after RA treatment are depicted in FIG. 8 byrealignment and enlargement of representative spots from the arraysdepicted in FIG. 7A, 7B, 7E, and 7F. For example, IGF2 mRNA wasdetectable only in total RNA and HuB mRNP complexes from RA treatedcells (FIG. 8). However, other HuB mRNP-bound mRNAs, such as integrinbeta, cyclin D2, and Hsp84 increased or decreased in abundancedisproportionately to their changes in the total RNA profile followingRA treatment (FIG. 8). The disparity between changes in the mRNAprofiles of total RNA and HuB mRNP complexes possibly results fromchanges in compartmentalization of mRNAs that flux dynamically throughmRNP complexes in response to RA treatment. In conclusion, the mRNAprofiles derived from these mRNP complexes are dynamic and can reflectthe state of growth, as well as changes in the cellular environment inresponse to a biological inducer such as retinoic acid.

Example 4 In vivo Target Sequence Preferences for RNA Binding Proteins

Using GenBank and EST databases, the 3′ UTR sequences from mRNAsenriched in RA-treated HuB mRNP complexes were identified (TABLE 2).Using the previously defined in vitro binding sequence for HuB, UUUAUUU,a Basic Local Alignment Search Tool (BLAST®) (National Center forBiotechnology Information (NCBI), Bethesda, Md.) analysis and/or visualinspection was performed to identify sequences similar to this consensusbinding site within the 3′ UTRs of neuronal HuB target mRNAsRecognizable HuB protein-RNA binding sequences were identified withinthe in vivo-captured mRNA subset. Many of the mRNAs for which 3′ UTRsequences were available contained similar uridylate-rich motifs tothose that bind to Hu protein in vitro. Moreover, most of these mRNAsencode proteins that are expressed in neuronal tissues or areup-regulated following RA-induced neuronal differentiation. The sequencealignment shown in TABLE 2 is consistent with the previous results wherein vitro selection was used to derive a consensus RNA binding sequencefor HuB. Using the methods described herein, it is possible to discernin vivo target sequence preferences for other RNA binding proteins.

TABLE 2 Gene 3′UTR Consensus Sequence CD44         UUUUCUAUUCCUUUUUUAUUU UAUGUCAUUUUUUUA IGF-2            UAAAAAACCAA UUUGAUU GGCUCUAAACA               UAAAGAA AUUAAUU GGCUAAAAACAUA                  CUAAAAUUAAUU GGCUUAAAAA HOX 2.5               UCACUCUU UAUUAUU AU                  AAAU UUUAUUA AGUUA                 AUCAGG UUCAUUUUGGUUGU Inhibitor                     AU UUUAUCU AGUUA J6   UUUUGUUUUUCUCCCUUUU UUCAUUU UGGUUGU GADD45     UAUUUUUUUCUUUUUUUUUUUUGGU CUUUAU       UUAAAUUCUCAGAAGU UUUAUUA UAAAUCUU Nexin 1        UUCUGUUAAAUAUU UUUAUAU ACUGCUUUCUUUUUU         AUUUUAUAGUAGUUUUUAUGU UUUUAUGAAAA              AUUUGCCUU UUUAAUU CUUUUU Egr-1           UAUUUUGUGGU UUUAUUU UACUUUGUACUU Zif268                     U UUUGUUU UCCUU Neuronal-                    UUUUUUAUUU UCUGUAUUUUUU Cadherin        UUUUUUUUAAAUUUU UUUAUUU UCUUUUU          UUUUUUAUUUUC UGUAUUU UUU             UUUUUAAUUU UUUAAUU UUUUUUIntegrin alpha                  AAUGG UUUAUAU UUAUGAU 5                   UUG UUUAUAU CUUCAAU SEF2              UUCAAGCGCUUGANUU Cf2r           UGCAUCGAUCCG UUGAUUU ACUACU Intefrin              UAUAAUUU UUAAUUU UUUAUUAUUUU beta UAUUUUACCUUUUUUUUUUUUCUUUAAUU CGUGGU CTCF             UUAUGAAUGU UAUAUUU GU                    UC UUAAUUU UUUCUCUUUUUUUUCU TGF beta 2          UUUUUUUUUCCU UUUAAUU GUAAAUGGUUCUUU  UUAAUGAUCAUUCAGAUUGUAUAUAUUU GUUUCCUUU             UUCAAUUUUU UUUAUAU ACUAUCUU              UUUUUC-- UUUAAUU GGUUUUUUMTP----       UGUCUUGUTCUGAGCAUUUAUUU UCAAA            UUCUCGUCUUG UUUAUUU UACAA           UAUAAUAAUAG UUUAUGU UUUGGAUGUUUGGU Cyclin D2         AUGUCUUGUUCUU UGUGUUU UUAGGAU                (AU/GA) UUUAUUU(UA/AG)

Example 5 Target Discovery Using Ribonomic Profiles

The steps for target discovery exemplified below are summarized in FIG.9.

Establishing the expression profiles of RNA binding protein genes. RNAbinding protein expression profiles were generated in normal anddiseased human tissues. Initial tissue and disease screening of RNAbinding proteins was accomplished by Quantitative ReverseTranscriptase-PCR using oligo dT-primers and commercially available RNAsamples (Stratagene, Inc., La Jolla, Calif.; Ambion, Inc., Austin, Tex.;BD Biosciences Clontech, Palo Alto, Calif.). 10-100 μg of cDNA was usedto perform Quantitative PCR using SybrGreen (Molecular Probes, Inc.,Eugene, Oreg.) and gene specific PCR primers on a BioRad iCyclerQuantitative PCR machine using protocols provided by the manufacturer.Experimental results were analyzed using the accompanying BioRad iCyclersoftware. RNA levels for candidate genes were normalized to rRNA.

For more rapid and comprehensive screening of tissues and cell lines, acustom RIBOCHIP™ spotted microarray (Ribonomics, Inc., Durham, N.C.) wasdesigned and manufactured under contract (MWG Biotech USA, Highpoint,N.C.). A gene list of known and putative human RNA binding protein geneswas compiled from a wide variety of public databases and search toolsincluding GenBank (NCBI, Bethesda, Md.), PubMed (NCBI, Bethesda, Md.),SRS Evolution (LION Biosciences, Cambridge, Mass.), LocusLink (NCBI,Bethesda, Md.), Protein FAMily database (pFAM); Welcome Institute SangerInstitute, Hinxton, UK), GO Database (Gene Ontology™ Consortium) andStructural Classification of Proteins (SCOPO) Package (Medical ResearchCouncil, Cambridge, UK). This array contained 50 mer oligonucleotides onglass slides corresponding to greater than about 1,400 RNA bindingproteins genes in duplicate, non-contiguous positions (plus controlgenes).

To screen for the expression of RNA binding proteins, RNA was preparedfrom cells in culture and from snap frozen clinical tissues according tothe Qiagen RNeasy® protocol (Qiagen, Inc., Valencia, Calif.). Total orpoly A+ RNA was labeled without amplification through generation of cDNAby reverse transcription in the presence of amino allyl-dUTP followed bydirect coupling to Cy3 or Cy5 fluorescent dyes (TIGR SOP#M0004).Hybridization and washing were performed by standard procedures (TIGRSOP#M0005). Data flow for data analysis and statistical analysis isshown in FIG. 10. In short, microarray slides were first scanned andread by a GENEPIX® Axon 4000B scanner using GENEPIX® 4.0 software (AxonInstruments, Inc., Union City, Calif.) for data acquisition. Spotfeatures are then extracted with Biodiscovery's IMAGENE™ V.4.2 package(BioDiscovery, Inc., Marina Del Rey, Calif.). Data preprocessing,including intra- and inter-array data normalization, centralization, andscaling, was accomplished through by visual (e.g., heat map) andquantitative methods (e.g., distribution analysis) implemented using thestatistical environment R (Ross Ihaka and Robert Gentleman, R: Alanguage for Data Analysis and Graphics, Journal of Computational andGraphical Statistics, 1996, 5, 299-314; hereby incorporated byreference) and BioConductor Suite of microarray data normalization andanalysis libraries (BioConductor, Biostatistics Unit of Dana FarberCancer Institute, Boston, Mass.). Final data analysis with normalizationand scaling was then accomplished using-gene clustering, statisticalfiltering and class prediction functions within the GENESPRING® 4.2.1software platform (Silicon Genetics, Redwood City, Calif.). Based uponarray data, RNA binding proteins that are up or down regulated (e.g.,differential RNA binding protein mRNA levels) to a statisticallysignificant extent in a tissue or disease specific manner were selectedfor confirmation studies by Quantitative PCR, Northern blot and Westernblot analyses.

Cloning and Expression of RNA Binding Protein Genes in BacterialVectors. As soon as candidate, differentially expressed RNA bindingproteins were identified, full length cDNA clones were generated byreverse transcriptase-PCR using commercial RNA tissue sources. Fulllength plasmid clones were constructed based on phage lambda-based (att)site-specific recombination protocols (Invitrogen, Corp., Carslbad,Calif.) for the GATEWAY™ pENTRD-Topo entry vectors and pDEST17 6×Hisdestination vectors (Invitrogen, Corp., Carslbad, Calif.). Escherichiacoli (e.g., BL21S1 or BL21A1) expressing polyhistidine-tagged RNABinding Protein fusion proteins were grown to mid-log phase at 37° C.and induced 0.3 M NaCl for BL21SI cells or 0.2% mM arabinose or 0.1 mMIPTG for BL21A1 cells at 20-37° C. for 2-6 hours (based uponoptimization in pilot expression studies for each clone). Bacterialcells were lysed by sonication and the fusion protein was purified onnickel columns (Qiagen, Inc., Valencia, Calif.) using standard methods.Insoluble fusion proteins were maintained and purified in the presenceof 8M urea, and soluble proteins were maintained in PBS. Purifiedrecombinant proteins were used for immunization of rabbits and/orchickens for production of polyclonal antibodies (typically throughcontract production). Polyclonal antibodies were characterized for theirability to immunoprecipitate and western blot native and recombinantproteins.

Interrogation of mRNP complexes. RNA binding proteins that are expressedin a tissue or disease specific manner are surrogate markers forcellular alterations due to the post-transcriptional processing offunctionally related groups of mRNAs. Changes in the abundance orconstellation of RNA binding proteins in a cell undoubtedly affect theprocessing of any mRNAs bound by those RNA binding proteins. The mRNAsassociated with these specific RNA binding proteins are very likely tobe critically or causally involved in the phenotype of the cell. Thus,as a subset, those genes whose mRNAs are associated with tissue ordisease specific mRNP complexes are a rich source of therapeutic targetsfor drug discovery.

Prioritized RNA binding proteins (e.g., RNA binding proteins thatexhibited the most dramatic variations with regard to expression)proceeded into the second stage of analysis, the Ribonomic AnalysisSystem (RAS™) assay (Ribonomics, Durham, N.C.) (FIG. 11). The RAS™ assayis the affinity isolation and characterization of in vivo formedcomplexes of mRNA and RNA binding proteins or mRNP complex-associatedproteins. Antibodies specific to the RNA binding proteins, mRNAcomplex-associated protein, or tags on RNA binding protein or mRNAcomplex-associated protein of interest were used to co-immunoprecipitatethe RNA binding protein and the associated subset of mRNAs according tothe manufacturers instructions. Using polyclonal or monoclonalantibodies raised to the selected RNA binding proteins, or to tags onthe RNA binding proteins or mRNA complex-associated proteins, mRNPcomplexes were isolated from cell or tissue lysates as described below,and optimized for each RNP complex. The extracted RNA was analyzed in astandard microarray format. Variations on the method have includedreversible chemical crosslinking with formaldehyde, use of a variety oftags and beaded reagents (cross-linked to synthetic beads, e.g.,sepharose), or inclusion of particular concentrations of salt (e.g.,NaCl or KCl) or detergent (e.g., NP-40, deoxycholate) based upon pilotstudies. For the analysis of mRNAs associated with mRNP complexes,commercially available glass slide arrays (e.g., such as Agilent HumanUnigene 14K (Agilent, Palo Alto, Calif.), MWG Pan Human 10K (MWGBiotech, Inc., High Point, N.C.), or membrane arrays, such as Atlas™Arrays (BD Biosciences, Clontech, Palo Alto, Calif.)), were utilizedusing protocols for hybridization, washing, and development provided bythe array manufacturers.

Preparation of Cell Free Extracts. Composition of the RAS™ assay lysisbuffer (RLB) may vary depending upon the binding characteristics of aparticular RNA Binding Protein for target RNAs Basic RLB contained 50 mMHEPES, pH 7-7.4, 1% NP-40, 150 mM NaCl, 1 mM DTT, 100 U/ml RNase OUT,0.2 mM PMSF, 1 μg/ml aprotinin and 1 ug/ml leupeptin. Variations ofthese basic components included changes in salt concentrations (0-500 mMNaCl or 0-5 mM KCl), ionic conditions (0-10 mM MgCl₂ or 0-20 mM EDTA),and reducing environment (0-5 mM DTT). For example, in order to preparecell extracts for examining the PTB mRNP, cultured cells were washed inice-cold PBS and scraped directly into RLB containing 5 mM MgCl₂ andincubated on ice for 10 minutes followed by centrifugation at 3,700×gfor 10 min at 4° C.

It is necessary in certain cases to crosslink the RNA binding protein totarget mRNAs prior to lysis and mRNP isolation. This was performed oncultured cells as well as fresh tissue samples. The extent ofcrosslinking was titrated for each cell line or tissue and monitoredbased on ability to immunoprecipitate mRNA in the complex. Culturedcells or tissue were incubated in PBS containing 0-1% formaldehyde atroom temperature for 15-60 min. Crosslinking was then quenched by theaddition of 1M Tris to a final concentration of 250 mM and incubatedfurther for an additional 20 minutes. The samples were then washed 3× inPBS containing 50 mM Tris. For cultured cells, the pellet wasresuspended in Radioimmunoprecipitation (RIPA) buffer (50 mM Hepes, pH7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC and 100 U/ml RNase Out)at approximately 2 mg/ml final protein concentration. For tissue, thesamples were resuspended in RIPA and homogenized with a Polytron todisrupt the tissue. Following the initial lysis, the samples (tissue andcultured cells) were subjected to sonication with a probe sonicator atoutput setting 6 (Branson 450, Branson Ultrasonics Corp., Danbury,Conn.) two times for 20 seconds each. Between sonications the sampleswere allowed to cool on ice for 2 minutes. Lysates were then cleared bycentrifugation at 3,700×g for 15 min. Subsequently, MRNP isolation asdescribed above was performed.

Immunoprecipitation of mRNP and RNA Extraction. On average, typicalfinal protein concentrations for the cellular lysates were 2 mg/ml.Approximately 2 mg were used for each immunoprecipitation condition. Thecleared cellular extracts were incubated with primary antibody (e.g.,anti-PTB was used at a final concentration of 10 μg/ml) or a controlantibody at equal concentration (e.g., pre-immune or IgG sera at finalconcentration of 10 μg/ml) for 2 hours at 4° C. A 25 μl aliquot ofProtein A Trisacryl (Pierce Biotechnology, Rockford, Ill.) was added andthe samples rotated for 1 hour at 4° C. The immune complex was thenwashed 6× in RLB buffer by adding 1 ml/wash of RLB buffer followed bybrief centrifugations in a microcentrifuge for 30 seconds at 5,000 rpm.After the final wash, 50 μl of RNA extraction buffer from the PicoPure™RNA isolation kit (Arcturus, Inc., Mountain View, Calif.) was added tothe beads, vortexed briefly and centrifuged to pellet the beads. Theextracted RNA was purified following the PicoPure™ protocol (Arcturus,Inc., Mountain View, Calif.). RNA present in the mRNP complex was thenquantified using the RiboGreen™ assay (Molecular Probes, Inc., Eugene,Oreg.).

Amplification of RNA, for Microarray Analysis. Since mRNA isolated frommRNP complexes represents only a small subset of total RNA, we amplifiedthe isolated mRNA prior to labeling. Message Amp™ (Ambion, Inc., Austin,Tex.) was used for RNA amplification according to the manufacturersinstructions. Two rounds of amplification were performed prior tolabeling by random primer polymerization with Cy3 or Cy5-dUTP.Hybridization and washing were performed according to the microarraymanufacturers' protocols and as described above. Microarray dataacquisition and analysis were performed as described above.

Analysis of mRNP Cluster Microarray Results. In a standard RAS™ analysis(e.g., normal vs. disease, treated vs. untreated), quantitative andqualitative changes in the total RNA content were compared to changes inthe mRNP complex. The data obtained was fractionated into four classes:(1) mRNP Transcripts that show comparable quantitative changes in themRNP complex, (2) RNAs present in the total RNA but not in the mRNPcomplex, (3) RNAs present in the mRNP complex but apparently absent orbelow the level of detection in the total, and (4) RNAs that change inthe cluster in a quantitatively different manner than in the total RNAanalysis. In addition, the RAS™ assay identifies genes represented byclass 4 that do not change in total abundance but that are repartitionedwithin the cell for alternative processing and regulation. As a result,different splice variants may be translated, the mRNA might betransported to and translated at a specific location within the cell, ortranslation itself might be up or down modulated. The subsets of genesidentified within groups 3 and 4 cannot readily be identified by anyother currently available approach to characterization of geneexpression. Analysis of mRNP complexes reveals mRNAs that are enrichedin the complex but otherwise present at sufficiently low levels to belost to background in the total RNA. For example, in a recentpublication looking at mRNAs associated with the Fragile X MentalRetardation Protein (FMRP) complex three genes (KIAA0561-like, Rab3GDP/GTP exchange protein, and hCT25324/(Celera, Rockville, Md.)) wereconsidered “absent” in the total RNA class and enriched 30-60 fold inthe FMRP complex class. Furthermore, this technique identifies genes ina disease or a cellular state whose expression is carefully controlledby the cell, and thus very likely to represent attractive targets fordrug discovery. Since one goal of the invention is the identification ofdrug targets, downstream efforts are focused on target classes that haveproven to be tractable targets for small molecule drugs. Specifically,these target classes include nuclear receptors, G-protein coupledreceptors, phosphodiesterases, kinases, proteases, and ion channels,among others. Other target classes of therapeutic interest includesecreted molecules, extracellular ligands, and phosphatases. Among thesegene classes, particularly attractive targets are those with the mostrestricted systemic expression profile.

Functional Verification of mRNP complexes. For candidate target genes orgene products identified by the RAS™ assay, expression at the RNA andprotein levels was confirmed by quantitative PCR and Western blot.Furthermore, the function of an mRNP complex as it relates to the fateof the associated mRNAs, such as stability, degradation or subcellularlocalization, was explored through a variety of techniques including,but not limited to, confocal microscopy, in situ hybridization, 3-hybridreporter analysis to confirm ternary interaction between mRNA and theRNA binding protein or mRNP complex-associated protein, and in vitromethods assessing biochemical activities. Such studies were supported bythe in vitro demonstration of RNA binding protein binding to specificnucleotide sequences typically found at the 5′ or 3′ end of the mRNA.

Validation of a Therapeutic Target's Role(s) in Disease or CellularPhenotype by RNAi. To confirm that a gene identified in a diseasespecific mRNP complex plays a direct role in the etiology of disease orother phenotype being studied, candidate target genes were chosen forRNAi inhibition studies. For each candidate therapeutic gene, one ormore short DNA segments representing the coding sequence of that genewas individually cloned into a plasmid vector in the sense or antisensedirection, downstream of a U6 polymerase III promoter or RNAse P RNA H1.Plasmid vectors were constructed that contain two or more short DNAsegments of five candidate therapeutic genes in the sense and antisensedirections, downstream of a U6 polymerase III promoter or RNAse P RNAH1. Alternatively, one may construct an RNAi by annealing chemicallysynthesized complementary 22 bp RNAs (Dharmacon, Lafayette, Colo.).Following transfection of the vector or double stranded RNA intocultured cells, phenotypic characteristics were evaluated to determinethe effect of inhibiting the expression of the candidate target. Inaddition, to verify inhibition of gene expression at the RNA and proteinlevels, Northern blots and Western blots of time course experiments wereperformed to demonstrate the efficacy and duration of inhibition for theindividual genes. Transfections can result in transient expression forone to five days. Alternatively, vectors expressing RNAi can be stablyexpressed in cultured cells by co-transfection and selection with adominant selectable marker such as neomycin. As alternatives to the useof RNAi, traditional antisense DNA or vectors expressing dominantnegative forms of targets of interest can be used. Antisense anddominant negative genes can be delivered by direct DNA transfection orthrough the use of virus vectors including, but not limited to,retroviruses, adenoviruses, adeno associated viruses, baculoviruses,poxviruses, and polyomaviruses. The biological system of study chosen todemonstrate the role of a gene in disease or cellular phenotype is basedupon knowledge in the art of the biological system, including a cellculture or animal model system, that mimics relevant biological features(e.g., uncontrolled growth of a tumor cell).

In addition to inhibition of the candidate target genes, RNAi constructswere use to inhibit expression of the RNA binding proteins that boundthe mRNAs of the candidate genes. Although inhibition of RNA bindingproteins that bind and regulate multiple mRNAs is likely to have a moredrastic effect on the phenotype of the cells, this procedure remains animportant control to verify the critical importance of the mRNP complex.

Example 6 Discovery and Validation of Novel Targets for NeuronalDifferentiation

HuB (Hel-N2) is an RNA binding protein that is believed to play a rolein regulating neuronal differentiation. The functional importance ofmRNAs in the HuB mRNP complex that are uniquely and critically involvedin the differentiation process of p19 cells, an embryonic carcinoma cellline, to a neuronal and glial-like phenotype were identified andvalidated. As a control, the experiments were duplicated by inducing p19cells to differentiate into a cardiac and skeletal muscle fate by DMSOtreatment.

Cell Culture. G11 cells, which are P 19 cells stably transfected with aSV40 promoter-driven pAlpha2-gene10-HuB plasmid that ectopicallyexpressed a g10-tagged neuron-specific HuB protein termed Hel-N2, wereobtained from Duke University (Durham, N.C.). The transfected plasmidwas maintained in the G11 cells by supplementing the medium with 0.2mg/ml G418 (Sigma Chemicals, St. Louis, Mo.). Parental, untransfectedmurine P19 cells were obtained from the American Type Culture Collection(ATCC) and maintained in monolayer culture using α-MEM without phenolred supplemented with 7.5% Bovine Calf Serum, 2.5% Fetal Bovine Serumand 100 U Penicillin/Streptomycin. Cells were grown in tissue cultureflasks or plates that had been pre-coated with 0.1% gelatin that wasremoved prior to use. Monolayer cell cultures were maintained in 5% CO₂at 37° C.

Neuronal differentiation was induced by treating 5×10⁵ G11 or P19 cellsplaced on a 60 mm petri dish with 0.5 μM RA (Sigma-Aldrich, St. Louis,Mo., Catalog #R2625). Muscle differentiation was induced by treating5×10⁵ p19 cells placed on a 60 mm petri dish with 5% DMSO(Sigma-Aldrich, St. Louis, Mo., Catalog #D2650). After two days, 25% ofthe cells that had formed into clumps were removed, placed in new petridishes, and supplemented with fresh medium and RA or DMSO. Following anadditional two days, cell aggregates were washed once withphosphate-buffered saline (PBS) and trypsinized. The cells were thenplated into two 100 mm gelatin-coated tissue culture plates. Cells wereharvested after an additional four days. The RA-treated G11 cells grewneurites and displayed characteristic neuronal markers and morphology,but did not terminally differentiate and remained susceptible to killingwith mitotic inhibitors. The DMSO treated G11 cells displayedcharacteristic muscle cell markers and morphology, but did notterminally differentiate and remained susceptible to killing withmitotic inhibitors. Cell extracts and immunoprecipitations wereperformed as described in Example 1. Monoclonal anti-gene 10 (g10)antibodies were obtained from Duke University (Durham, N.C.) aspreviously described in Examples 1 and 2.

Following immunoprecipitation, the associated mRNAs were identified onhigh density mouse 12K glass slide microarrays (Mouse cDNA microarraykit, Catalog #G4104A, Agilent Technologies, Palo Alto, Calif.). RNAlabeling and microarray analysis was performed according to themanufacturer's protocols (Agilent Technologies, Palo Alto, Calif.).Comparison of the mRNA profiles before and after RA treatment revealedthat, in contrast to the approximately 10K genes that were expressed inthe total RNA samples, only 1,072 genes were expressed in the G11 HuBmRNP after RA treatment. Further, when the 1,072 genes were broken downinto drug-treatable gene classes (e.g., protein classes to whichconventional drug therapies are targeted) based on GO (Gene Ontology™Database) classifications there were 27 kinases (compared to 275detected in total RNA), 43 phosphatases (compared to 148 detected intotal RNA), 14 Proteases (compared to 137 detected in total RNA), 14Receptors (compared to 87 detected in total RNA), 39 cytokines (comparedto 143 detected in Total RNA), and 20 Growth Factors (compared to 87detected in total RNA) (TABLE 3).

TABLE 3 # of Genes Detected in # of Genes Detected in Gene Class TotalCellular RNA HuB RNP Complex Kinases 275 27 Phosphatases 148 43Proteases 137 14 Receptors 87 14 Cytokines 143 39 Growth Factors 87 20

TABLE 4 Gene ΔHuB RNP* ΔTotal Cellular RNA* CACN +14.53X +1.31X CELSR+9.41X −1.33X MBNL +8.95X +1.42X *Change in amount of RNA for each genein the HuB RNP or in the total cellular RNA comparingdifferentiated/undifferentiated in G11 cells at day 1 and day 9.

From five repeat immunoprecipitations, HuB mRNP complex—associated mRNAswere selected that were reproducibly found to be selectively localizedto the HuB mRNP complexes with or without substantial changes in thetotal RNA content in the G11 cells. Among the top 40 genes sorted bylargest quantitative increase in the HuB complex, 19 were expressedsequence tags (EST) or Riken clones for which little or no biologicalinformation is currently available. Additionally, of those 40, 14 wereincreased greater than 2× in both the HuB mRNP complex and in the totalRNA. Three specific genes were chosen for subsequent analysis: calciumchannel beta 3 subunit (CACN), cadherin EGF LAG seven-pass G-typereceptor (CELSR), and muscle blind RNA binding protein gene (MBNL)(TABLE 4). The changes in mRNA levels in the HuB mRNP compared tochanges observed in the total amount of mRNA in the cell for the threegenes are shown in TABLE 4.

To verify a role in neuronal differentiation, a comprehensivetranscriptional analysis by Quantitative PCR was undertaken looking atthe patterns of RNA expression for the CACN, CELSR, and MBNL genes inthe G11 cells (constitutively expressing HuB) and in the parental P19cells (FIGS. 12A, 12B, and 12C). For the test cell line, G11, all threegenes showed temporal induction over the time course of differentiationfollowing treatment with retinoic acid. In contrast, DMSO induction ofG11 cells failed to induce changes in the expression patterns of thesame genes, suggesting that, although these genes play a role inneuronal differentiation, expression of these genes is not required formuscle differentiation.

To further confirm a role for these three genes in neuronaldifferentiation, expression was examined by Quantitative PCR in the P19parental cell line lacking the ectopically expressed g10-tagged HuB(FIGS. 13A, 13B, and 13C). Although with slightly varied kinetics, theexpression of all three genes is altered over the course ofdifferentiation to neurons, but remains relatively unchanged duringdifferentiation to muscle.

Thus, in the model system of G11 cells, a simple microarray analysisusing total RNA samples derived from a comparison of days one and ninewould fail to highlight the importance of these three genes in neuronaldevelopment. These data are confirmed by the Quantitative PCR levelsseen for the G11 cells when comparing day one and day nine samples.However, since they were re-assorted into HuB-containing mRNPs, andsince HuB is known to be involved in neuronal differentiation, they wereimplicated in neuronal differentiation. Such a role is confirmed by thesingle or dual phase differential expression patterns observed for thethree genes between days one and nine in both the test cell line, G11,and more importantly, in the parental cell line, P19. Thus, analysis ofcritical mRNPs can readily highlight genes that are important inparticular biological processes.

Example 7 Screening of RNA Binding Proteins as Sentinels of Hepatoxicity

The human cell line HepG2 was used as a model for hepatotoxicity. HepG2cells were treated with test compound doses and effects on RNA bindingprotein gene expression were assessed. Doses that produced 50% mortality72 hours post-treatment were used to treat HepG2 cells for 24 hours, atwhich time the effects on gene expression were determined. By using a 24hour time point for assessing gene expression, it was possible toexamine changes in gene expression elicited by compound doses that leadto significant cell death.

Preliminary dose response curves were generated to determine a HighestTolerated Dose (HTD), the concentration of test compound that producedthe minimum detectable morphological changes in the cells (e.g.,rounding, vesiculation, detachment, lysis). Briefly, cells were seededinto 96 well tissue culture plates at 2×10⁴ cells/well in Dulbecco'sModified Eagle's Medium (DMEM) with 10% FCS. Twenty-four hours afterseeding, the media was removed and fresh DMEM containing 0.1% BSAwithout FCS and either 0.25% Dimethyl Sulfoxide (DMSO) or 0.25% DMSO, inaddition to dilutions of test compound, were added to the cells. Forexample, dilutions ranging from 4 μM to 10 mM were used for thecompounds Clofibrate, DEHP, Gemfibrozil, Phenyloin, and Acetaminophen(Sigma-Aldrich, St. Louis, Mo.).

Twenty four hours after dosing, the cells were assessed visually formorphological changes. The HTD was used to define a narrower dose rangeto be used in a vital dye cell viability assay (e.g., Alamar Blue, MTT(Roche Applied Science, Indianapolis, Ind.), XTT (Roche Applied Science,Indianapolis, Ind.)). For the vital dye assays, cells were seeded anddosed as described above for 72 hours rather than 24 hours. The 72 hourvital dye toxicity assessment data was used to determine TD₅₀ values foreach compound, i.e., the Toxic Dose producing about 50% cell deathrelative to DMSO treatment alone.

The concentration of test compound producing 50% cell death following a72 hour cell treatment was used to dose cells for mRNA analysis. Cellswere seeded into T150 plates at about 20% confluency (equivalent densityto that used in TD₅₀ determinations) in DMEM with 10% FCS for 24 hours.The medium was removed and replaced with fresh medium containing DMEMand 0.1% BSA (i.e., without FCS) and DMSO alone or test compound andDMSO at the determined TD₅₀ concentration. Cells were harvested 24 hoursafter dosing. Total RNA was extracted from fresh or snap frozen cellpellets using the Qiagen RNeasy protocol (Qiagen, Inc., Valencia,Calif.). Total RNA was quantified by spectroscopy. Integrity wasverified by separation on the Agilent Bioanalyzer 2100 (Agilent, PaloAlto, Calif.). Total RNA was labeled without amplification throughgeneration of cDNA by reverse transcription in the presence of aminoallyl-dUTP followed by direct coupling to Cy3 or Cy5 fluorescent dyes(TIGR SOP#M0004). The labeled RNA was analyzed using a custom spottedoligonucleotide microarray containing approximately 1400 RNA bindingproteins (MWG Biotech, High Point, N.C.). For initial screening, aRiboChip™ V. 1.0 microarray (Ribonomics, Durham, N.C.) was used. TheRiboChip™ (Ribonomics, Durham, N.C.) was composed of oligonucleotidescomplementary to greater than 1400 RNA Binding Protein genes pluscontrols. Alternatively a RiboChip™ microarray (Ribonomics, Durham,N.C.) may also include a comprehensive collection of transcriptionfactors to represent the full set of sentinel genes for the quantitativeand qualitative assessment of toxicity. Hybridization of labeled probeand washing were by standard procedure (TIGR SOP#0005).

Data flow for data analysis and statistical analysis is shown in FIG.15. In short, microarray slides were first scanned and read by aGENEPIX® Axon 4000B scanner using GENEPIX® 4.0 software (AxonInstruments, Inc., Union City, Calif.) for data acquisition. Spotfeatures are then extracted with Biodiscovery's IMAGENE™ V.4.2 package(BioDiscovery, Inc., Marina Del Rey, Calif.). Data preprocessing,including intra- and inter-array data normalization, centralization, andscaling, was accomplished through by visual (e.g., heat map) andquantitative methods (e.g., distribution analysis) implemented using thestatistical environment R (Ross Ihaka and Robert Gentleman, R: Alanguage for Data Analysis and Graphics, Journal of Computational andGraphical Statistics, 1996, 5, 299-314; hereby incorporated byreference) and BioConductor Suite of microarray data normalization andanalysis libraries (BioConductor, Biostatistics Unit of Dana FarberCancer Institute, Boston, Mass.). Final data analysis with normalizationand scaling was then accomplished using gene clustering, statisticalfiltering and class prediction functions within the GENESPRING® 4.2.1software platform (Silicon Genetics, Redwood City, Calif.) to identifyhighly predictive gene sets unique to individual compounds, compoundclasses, and general toxic responses.

These predictive gene sets served as the basis by which to assesschanges in gene expression profiles of HepG2 cells elicited by unknowntest compounds and as a means to predict and classify hepatotoxicitycaused by these compounds (Example 9). In addition, these data are usedto predict toxicity in other tissues. Changes in gene expression inHepG2 cells in response to exposure to a test compound were compared toan internal database of gene expression information for a largecollection of normal tissues. RNA binding protein genes with expressionpatterns that are perturbed upon test compound treatment in HepG2 cellsare used to predict toxicity of the compound tissues that express thatRNA Binding Protein. For example, if a compound elicits a change inexpression of a gene uniquely expressed in normal cardiac tissue, thenthe compound are flagged as having potential for cardiac toxicity.

Example 8 Determining the Mechanism of Toxicity or Mechanism of Actionof a Compound by Characterizing mRNP Complexes

To validate the mechanistic connections between altered transcriptionalregulators and downstream phenotypic effects, the identification oftranscription factors and RNA Binding Proteins whose expression isconsistently altered in the presence of specific toxicants lead to theidentification of downstream genes affected by changes in thesetranscriptional regulators.

One of the most valuable components associated with the use oftranscription factors and RNA binding proteins as sentinels of toxicityderives from their subsequent value for providing mechanistic insightsand studies (FIG. 16). The consistent up-regulation of a transcriptionfactor or RNA binding protein by a toxicant is very likely to result indownstream effects on genes regulated by these regulators. Such genescan be provisionally identified due to the occurrence of transcriptionfactor binding sites in cis regulatory elements upstream of the codingregion of the gene. The DNA binding activity of individual transcriptionfactors can be readily evaluated in toxicant treated cell lysates by invitro gel retardation assays which measure the ability of a protein tobind to a target DNA sequence and retard its migration during gelelectrophoresis. Alternatively, chromatin immunoprecipitation coupled tomicroarray analysis may be used to identify transcription factor boundsegments of DNA. Functional assessment of the regulatory effects oftranscription factors is routinely accomplished with the use of reportergene assays in which one or more copies of the transcription factorbinding site is inserted upstream of a reporter gene, typically aforeign enzyme (e.g., β-galactosidase, chloramphenicol acetyltransferase, or a fluorescent molecule such as green fluorescent protein(GFP)). In these assays, changes in gene expression are quantitativelyreported through changes in enzymatic activity or fluorescent intensity.Thus, identification of toxicant-regulated transcription factors can bereadily assessed through the use of relatively standard laboratorytechniques to downstream, mechanistic effects on collections of cellulargenes.

The role of RNA binding proteins in the collection, organization, andcoordinate processing of associated mRNAs is potentially a rich sourceof information about downstream toxic effects mediated throughparticular genes. Since RNA binding proteins bind to conserved sequenceelements in the 5′ and 3′ UTRs of genes to regulate stability,transport, translation, and modulation of the expression of the RNAbinding proteins genes themselves, they will have effects on multipledownstream genes. For example, the RNA binding protein tristetraprolin(TTP) binds to AU-rich elements in the 3′UTR of TNF-α and GM-CSFresulting in accelerated degradation of those mRNAs. Furthermore, micedeficient in TTP develop severe pathology consistent with broadautoimmune disease including arthritis, dermatitis, mycloid hyperplasia,and cachexia, symptoms that can be abated by neutralization withantibodies to TNF-α. In a somewhat similar fashion, the congenitalabsence of another RNA binding protein, FMRP is the primary cause offragile X syndrome in humans, the most prevalent form of hereditarymental retardation. FMRP binds and localizes a subset of mRNAs to theneuronal synapse to enable localized protein translation in response toneurotransmitters. The absence of the FMRP is thought to causederegulation of a collection of mRNAs critical for synaptictransmission.

By defining RNA binding protein genes demonstrably up-regulated by aclass of compounds, the mRNA pools associated with these RNA bindingproteins can be identified. RNA binding protein genes that are regulatedby a class of compounds can be cloned and expressed as GST or 6×Histagged fusion proteins using standard cloning procedures and commercialexpression vectors. Using bacterial expression vectors, the RNA bindingproteins can be expressed either in E. coli or in coupled in vitrotranscription translation systems. Following incubation and attachmentof the recombinant proteins to nickel or GST beads that specificallybind the GST or 6×His tagged proteins, total RNA preparations fromcompound-treated cells can be added to the RNA binding protein: beads topermit binding of mRNAs from the HepG2 cells. In this manner, thosecellular mRNAs which are likely to be associated with the regulated RNAbinding protein in liver cells, and likely to be aberrantly affected bydrug treatment, can be identified by standard microarray analysis asdescribed above.

Alternatively, depending on the availability of antibodies specific tothe RNA binding proteins modulated by drug treatment, endogenous mRNPcomplexes from HepG2 cells can be immunoprecipitated and the mRNAsubsets interrogated as described above. The use of RNA binding proteinsaltered by compounds will identify pools of mRNAs that could bedifferentially and coordinately regulated in some aspect of splicing,nuclear transport, stability, subcellular localization, or translationof groups of genes with common functions important for the mechanism ofaction of a compound class.

Example 9 Isolation of Discrete mRNP Complexes from Cells and Tissues inOrder to Identify the Full Set of Associated RNA Binding Proteins andRNA Associated Proteins with an mRNA of Interest

An alternative and highly directed method called the RiboTrap™ assay(Ribonomics, Durham, N.C.) is used to detect RNA binding proteins andmRNP complex-associated proteins that are associated endogenously withdisease-related mRNAs in vivo. The assay defines the constellation ofmRNAs that are co-regulated with a gene of interest, such as a drugtarget. The information obtained can provide novel pathway informationfor validated targets, additional therapeutic targets for alternative ormulti-drug therapies, and surrogate disease markers for monitoring inclinical trials.

Using standard recombinant DNA and PCR technologies, a cDNA representingthe gene of interest and/or its 5′ and 3′ UTRs is constructed. The 3′UTR of the cDNA has a series of repeats of the stem-loop representingthe RNA binding site of the phage MS2 coat protein (FIG. 16).Alternatively, any RNA stem loop or other RNA structure that is known tobind a specific protein (e.g., HIV RRE and the Rev protein) could beused.

For prototype experiments, a c-myc cDNA is used because thecorresponding mRNA is a binding target of ELAV/Hu proteins both in vitroand in vivo. The c-myc cDNA is cloned into an expression vectorpossessing an appropriate mammalian cell promoter such as CMV, SV40 oractin promoters, or alternatively an adenovirus or retrovirus vector,and transfected into compatible mammalian cell line. For example, thecDNA encoding a neuronal protein is expressed in a neuronal cell linesuch as PC12 (rat), P19 (mouse), or hNT2 (human). Alternatively, for ametabolic study, the cDNA is expressed in a preadipocyte (mouse 3T3L1)or a human adipocyte line.

Following expression of the engineered c-myc cDNA, a cell extract isprepared to fish out the c-myc mRNA containing the MS2 coatprotein-binding site. MS2 coat protein is linked to agarose or Sepharosebeads or another suitable solid matrix. Antibody to MS2 coat protein orbiotinylated MS2 coat protein may also bind to Strepavidin beads. Thesereagents allow isolation from the cell extract of the mRNA containingthe MS2 stem-loop repeats and the RNA binding proteins and/or mRNAcomplex-associated proteins that are associated with the mRNA in vivo.

Variations on the method include chemical crosslinking with formaldehydeor the use of a variety of tags and beaded reagents. Proteins that areisolated in association with the mRNA of interest using the RiboTrap™assay (Ribonomics, Durham, N.C.) can be identified using standardproteomic methods. For example, Matrix Assisted LaserDesorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI TOF) andTandem Mass Spectrometry (or Mass Spectrometry/Mass Spectrometry(MS/MS)) can be used to identify peptide sequences for databasesearches. Antibodies reactive with identified proteins can be raisedaccording to standard methods and used to perform the RAS™ assay asdescribed previously.

Following application of the RAS™ assay (Ribonomics, Durham, N.C.), thesubpopulation of mRNAs that are present in mRNP complexes can beidentified and examined for the presence of common UTR sequenceelements. It should be noted that computational analysis for homology isnot a reliable method for identifying Untranslated Sequence Elements forRegulation Codes (USER codes) because they are often structural ratherthan single stranded. More importantly, the subpopulation of mRNAs canbe examined for functional relationships. For example, each mRNA can becategorized by gene annotation and by known functions in functionalgenomics databases (e.g., Locus Link (NCBI, Bethesda, Md.), GO Database(Gene Ontology™ Consortium), Proteome BioKnowledge® Library (IncyteGenomics, Inc., Palo Alto, Calif.)). For example, if the protein used inthe RiboTrap™ assay (Ribonomics, Durham, N.C.) is involved in immuneregulation, the other mRNAs found in the same mRNP complex can beanalyzed for their role in immune regulation. However, the mRNA could bebound indirectly through a different RNA binding protein in the mRNPcomplex (e.g., is assessed to the presence of USER code element in itsUTR that recognizes the RNA binding protein or other known binding sitesfor RNA binding proteins.

The goal of the RAS™ assay is to identify mRNA populations in which themRNAs have related structural features in their UTRs or the proteinsencoded by the mRNAs have functional relationships. Among the relatedfunctions that are expected are a) involvement of encoded proteins in acommon metabolic pathway, b) encoded proteins that are temporallyco-regulated, c) encoded proteins that are similarly localized in or onthe cell, d) encoded proteins that play a role in forming or regulatinga biological machine (e.g., a ribosome). The identification of complextraits and phenotypes that result from the expression of a set offunctionally-related proteins would include such processes as cognition,cell-specific activation, inflammation, or differentiation. Whileproteins known to be involved in these complex processes are known fromother studies, the majority of the functions remain largely unknown. Oneof the values of the invention is for discovering a larger set ofproteins involved in these processes that could serve as alternativedrug targets or surrogate markers.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore illustrative rather than limiting of theinvention described herein. The invention is described by the followingclaims, with equivalents of the claims to be included therein.

1. A method of evaluating an effect of a test compound on a cell, themethod comprising: a) contacting the cell with the test compound; b)lysing the cell to produce a lysate; c) isolating an mRNA-protein (mRNP)complexes from the lysate; d) identifying RNAs en masse from the mRNPcomplexes; f) comparing amounts of the RNAs identified in d) to those ina control, thereby evaluating the effect of the test compound on thecell.